Cell culture medium

ABSTRACT

Provided herein, inter alia, are compositions and methods for culturing mammalian cells. In certain aspects, the composition is a medium containing one or more of a lithium ion source, one or more fatty acids, and/or ethanol. Use of any of the cell culture media described herein to culture cells that have been genetically engineered to produce one or more recombinant polypeptides (for example, antibodies) can result in increased titers, a more favorable glycosylation profile, and/or modulated (e.g. decreased) amounts of high and low molecular weight species, and/or modulated (e.g. decreased) amounts of acidic or basic charge variants, compared to cells cultured in a medium that does not contain one or more of a lithium ion source, one or more fatty acids, and/or ethanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/498,221, filed on Apr. 26, 2017, which claims priority toU.S. Provisional Patent Application No. 62/327,964, filed on Apr. 26,2016. The disclosure of each of the above-referenced applications areherein expressly incorporated by reference in their entireties.

FIELD OF INVENTION

The invention relates generally to the field of cell culture media andmethods for using the same to produce useful cultured cell-derivedproducts.

BACKGROUND

Commercial production of cell culture-derived products (for example,protein-based products, such as monoclonal antibodies (mAbs)), requiresoptimization of cell culture parameters in order for the cells toproduce enough product to meet clinical and commercial demands. However,when cell culture parameters are optimized for improving productivity ofa protein product, it is also necessary to maintain desired qualityspecifications of the product such as glycosylation profile, aggregatelevels, charge heterogeneity, and amino acid sequence integrity (Li, etal., 2010, mAbs., 2(5):466-477).

For instance, an increase of over 20% volumetric titer results in asignificant improvement in large-scale monoclonal antibody productioneconomics. Additionally, the ability to control the glycan forms ofproteins produced in cell culture is important. Glycan species have beenshown to significantly influence pharmacokinetics (PK) andpharmacodynamics (PD) of therapeutic proteins such as mAbs. Moreover,the ability to modulate the relative percentage of various glycanspecies can have drastic results over the behavior of a protein in vivo.For example, increased mannose-5-N-acetylglycosamine-2 (“Man5”) andother high-mannose glycan species have been shown to decrease mAb invivo half-life (Liu, 2015, J Pharm Sci 104(6):1866-84; Goetze et al.,2011, Glycobiology, 21(7):949-59; and Kanda et al. 2007, Glycobiology,17(1):104-18). On the other hand, glycosylated mAbs withmannose-3-N-acetylglycosamine-4 (“G0”) glycan species have been shown toimpact antibody dependent cellular cytotoxicity (ADCC).

Bioreactors have been successfully utilized for the cell-basedproduction of therapeutic proteins using fed-batch, immobilized,perfusion and continuous modes. Strategies, such as the use oftemperature, media formulation, including the addition of growthinhibitors, autocrine factors or cyclic mononucleotides, andhyperstimulation by osmolarity stress, have been used to enhance proteinproduction by cells in culture. To the extent that they have worked atall, these approaches have shown only marginal success.

As such, there is a particular need for improved compositions for use incell culture for the production of medically or industrially usefulproducts, such as antibodies. Ideally, such compositions and methods forutilizing the same would result in higher titers, modulated (e.g.decreased) high and low molecular weight species, as well as a morefavorable glycosylation profile of the derived products in cell culture.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles, electronic databaseentries, etc.) are referenced. The disclosure of all patents, patentapplications, and other publications cited herein are herebyincorporated by reference in their entirety for all purposes.

SUMMARY

The invention provided herein discloses, inter alia, compositions andmethods for culturing mammalian cells. In certain aspects, thecomposition is a medium containing one or more of a lithium ion source,one or more fatty acids, and/or ethanol. Use of any of the cell culturemedia described herein to culture cells that have been geneticallyengineered to produce one or more recombinant polypeptides (for example,antibodies) can result in increased titers, a modulated glycosylationprofile, modulated amounts of acidic or basic charge species, and/ormodulated amounts of high and low molecular weight species compared tousing media that do not contain one or more of a lithium ion source, oneor more fatty acids, and/or ethanol.

Accordingly, in some aspects, provided herein is a medium for culturingmammalian cells comprising: (a) (i) a basal medium or (ii) a feedmedium; and (b) one or more sources of lithium ions. In someembodiments, the cells have been genetically engineered to produce oneor more recombinant polypeptides. In some embodiments of any of theembodiments disclosed herein, the medium further comprises (c) ethanol;and/or (d) one or more fatty acids. In some embodiments, said one ormore fatty acids is selected from the group consisting of oleic acid,linoleic acid, linolenic acid, myristic acid, palmitic acid and stearicacid. In some embodiments of any of the embodiments disclosed herein,said one or more sources of lithium ions is selected from the group ofone or more of lithium acetate, lithium chloride, lithium carbonate,lithium oxybutyrate, lithium orotate, lithium bromide, lithium citrate,lithium fluoride, lithium iodide, lithium nitrate, and lithium sulfate.In some embodiments of any of the embodiments disclosed herein, saidlithium ions are present in a concentration from about 0.1 μM to about25 mM. In some embodiments of any of the embodiments disclosed herein,the titer of said one or more recombinant polypeptides is increasedcompared to the titer of recombinant polypeptides produced by mammaliancells that are not cultured in said medium. In some embodiments of anyof the embodiments disclosed herein, the amount of high molecular weightspecies of said one or more recombinant polypeptides produced by saidcells is modulated (e.g. decreased) compared to recombinant polypeptidesproduced by mammalian cells that are not cultured in said medium. Insome embodiments of any of the embodiments disclosed herein, the amountof low molecular weight species of said one or more recombinantpolypeptides produced by said cells is modulated (e.g. decreased)compared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments of any of theembodiments disclosed herein, the glycosylation profile (e.g., theamount of glycosylation) of said one or more recombinant polypeptidesproduced by said cells is modulated compared to recombinant polypeptidesproduced by mammalian cells that are not cultured in said medium. Insome embodiments, said modulated glycosylation comprises modulated(e.g., decreased) terminal mannose glycan species. In some embodiments,said modulated glycosylation comprises modulation of one or more glycanspecies selected from mannose-5-N-acetylglycosamine-2 (Man5),mannose-6-N-acetylglycosamine-2 (Man6), mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), ormannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F). In someembodiments of any of the embodiments disclosed herein, the amount ofacidic or basic charge variants of said one or more recombinantpolypeptides produced by said cells is modulated (e.g., decreased)compared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments of any of theembodiments disclosed herein, ethanol is present at a concentration fromabout 0.001% to about 4% (v/v). In some embodiments of any of theembodiments disclosed herein, the one or more fatty acids are present ata concentration (such as a daily feeding concentration) of about 1 μM toabout 4 mM (such as any of about 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, or 4 mM, inclusive of all valuesfalling in between these numbers).

In other aspects, provided herein is a medium for culturing mammaliancells comprising: (a) (i) a basal medium or (ii) a feed medium; and (b)ethanol. In some embodiments, the cells have been genetically engineeredto produce one or more recombinant polypeptides. In some embodiments ofany of the embodiments disclosed herein, the medium further comprises(c) one or more fatty acids. In some embodiments, said one or more fattyacids is selected from Butyric (C4), Valeric (C5), Caproic (C6),Enanthic (C7), Caprylic (C8), Pelargonic (C9), Capric (C10), Undecylic(C11), Lauric (C12), Tridecylic (C13), Myristic (C14), Pentadecanoic(C15), Palmitic (C16), Margaric (C17), Stearic (C18), Nonadecylic (C19),Arachidic (C20), Heneicosylic (C21), Behenic (C22), Tricosylic (C23),Lignoceric (C24), Pentacosylic (C25), Cerotic (C26), Heptacosylic (C27),Montanic (C28), Nonacosylic (C29), Melissic (C30), Hentriacontylic(C31), Lacceroic (C32), Psyllic (C33), Geddic (C34), Ceroplastic (C35),Hexatriacontylic (C36), Heptatriacontanoic (C37), or Octatriacontanoic(C38) acids. In some embodiments of any of the embodiments disclosedherein, ethanol is present at a concentration from about 0.001% to about4% (v/v). In some embodiments of any of the embodiments disclosedherein, the titer of said one or more recombinant polypeptides isincreased compared to the titer of recombinant polypeptides produced bymammalian cells that are not cultured in said medium. In someembodiments of any of the embodiments disclosed herein, the amount ofhigh molecular weight species of said one or more recombinantpolypeptides produced by said cells is modulated (e.g. decreased)compared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments of any of theembodiments disclosed herein, the amount of low molecular weight speciesof said one or more recombinant polypeptides produced by said cells ismodulated (e.g. decreased) compared to recombinant polypeptides producedby mammalian cells that are not cultured in said medium. In someembodiments of any of the embodiments disclosed herein, theglycosylation profile (e.g., the amount of glycosylation) of said one ormore recombinant polypeptides produced by said cells is modulatedcompared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments, said modulatedglycosylation comprises modulated (e.g., decreased) terminal mannoseglycan species. In some embodiments, modulated glycosylation comprisesmodulation of one or more glycan species selected frommannose-5-N-acetylglycosamine-2 (Man5), mannose-6-N-acetylglycosamine-2(Man6), mannose-3-N-acetylglucosamine-4 (G0),mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), ormannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F). In someembodiments of any of the embodiments disclosed herein, the amount ofacidic or basic charge variants of said one or more recombinantpolypeptides produced by said cells is modulated (e.g., decreased)compared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments of any of theembodiments disclosed herein, the one or more fatty acids are present ata concentration (such as a daily feeding concentration) of about 1 μM toabout 4 mM (such as any of about 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, or 4 mM, inclusive of all valuesfalling in between these numbers).

In further aspects, provided herein is a medium for culturing mammaliancells comprising: (a) (i) a basal medium or (ii) a feed medium; and (b)one or more fatty acids. In some embodiments, the cells have beengenetically engineered to produce one or more recombinant polypeptides.In some embodiments of any of the embodiments disclosed herein, the oneor more fatty acids are present at a daily feeding concentration ofabout 1 μm-1 mM. In some embodiments of any of the embodiments disclosedherein, the amount of high molecular weight species of said one or morerecombinant polypeptides produced by said cells is modulated (e.g.decreased) compared to recombinant polypeptides produced by mammaliancells that are not cultured in said medium. In some embodiments of anyof the embodiments disclosed herein, the amount of low molecular weightspecies of said one or more recombinant polypeptides produced by saidcells is modulated (e.g. decreased) compared to recombinant polypeptidesproduced by mammalian cells that are not cultured in said medium. Insome embodiments of any of the embodiments disclosed herein, theglycosylation profile (e.g., the amount of glycosylation) of said one ormore recombinant polypeptides produced by said cells is modulatedcompared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments, said modulatedglycosylation comprises modulated (e.g., decreased) terminal mannoseglycan species. In some embodiments, modulated glycosylation comprisesmodulation of one or more glycan species selected frommannose-5-N-acetylglycosamine-2 (Man5), mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), ormannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F). In someembodiments of any of the embodiments disclosed herein, the amount ofacidic or basic charge variants of said one or more recombinantpolypeptides produced by said cells is modulated (e.g., decreased)compared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium. In some embodiments of any of theembodiments disclosed herein, the glycosylation profile of said one ormore recombinant polypeptides is modulated (e.g., altered) compared tomammalian cells that are not cultured in said medium. In someembodiments of any of the embodiments disclosed herein, the fatty acidis one or more of thymol, cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid,cholesterol, palmitic acid, stearic acid, and/or myristic acid.

In yet other aspects, provided herein is a method for producing one ormore recombinant polypeptides from an engineered mammalian cell, themethod comprising: (a) culturing said engineered mammalian cell in anyof the cell culture media disclosed herein under suitable conditions forthe production of said one or more recombinant polypeptides; and (b)producing said one or more recombinant polypeptides. In someembodiments, the method further comprises (c) isolating said one or morerecombinant polypeptides. In some embodiments of any of the embodimentsdisclosed herein, the medium is a basal medium. In some embodiments ofany of the embodiments disclosed herein, the medium is a feed medium. Insome embodiments of any of the embodiments disclosed herein, said one ormore recombinant polypeptides is an antibody or fragment thereof. Insome embodiments, said antibody is a monoclonal antibody. In someembodiments, the monoclonal antibody inhibits the growth of aproliferating cell. In some embodiments, said antibody or fragmentthereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25,CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa,IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF),CD19, CD30, interleukin-1 beta (IL1(3), prostate specific membraneantigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertasesubtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₂,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7. In some embodiments, said monoclonal antibody istrastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab,ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab,efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.

In another aspect, provided herein are methods for modulating theglycosylation profile of one or more recombinant polypeptides producedby a genetically engineered mammalian cell, the method comprising: (a)culturing said mammalian cell in any of the cell culture media disclosedherein under suitable conditions for the production of said one or morerecombinant polypeptides; and (b) producing said one or more recombinantpolypeptides, wherein said one or more recombinant polypeptides has amodulated glycosylation profile compared to recombinant polypeptidesproduced by mammalian cells that are not cultured in any of the cellculture media disclosed herein. In some embodiments, said modulatedglycosylation profile comprises modulated (e.g., decreased) terminalmannose glycan species. In some embodiments, said modulatedglycosylation comprises modulation of one or more glycan speciesselected from mannose-5-N-acetylglycosamine-2 (Man5),mannose-6-N-acetylglycosamine-2 (Man6), mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), and/ormannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F). In someembodiments of any of the embodiments disclosed herein, the ratio of theterminal mannose glycan species to the total sum of glycan species ismodulated (e.g., decreased) by about 40% to about 50%. In someembodiments of any of the embodiments disclosed herein, said one or morerecombinant polypeptides is an antibody or fragment thereof. In someembodiments, said antibody is a monoclonal antibody. In someembodiments, the monoclonal antibody inhibits the growth of aproliferating cell. In some embodiments, said antibody or fragmentthereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25,CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa,IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF),CD19, CD30, interleukin-1 beta (IL1(3), prostate specific membraneantigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertasesubtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7. In some embodiments, the monoclonal antibody istrastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab,ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab,efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.

In another aspect, provided herein are methods for modulating (e.g.,reducing) the amount of high or low molecular weight species of one ormore recombinant polypeptides produced by an engineered mammalian cell,the method comprising: (a) culturing said mammalian cell in any of thecell culture media disclosed herein under suitable conditions for theproduction of said one or more recombinant polypeptides; and (b)producing said one or more recombinant polypeptides, wherein said one ormore recombinant polypeptides have reduced amounts of high or lowmolecular weight species compared to recombinant polypeptides producedby mammalian cells that are not cultured in any of the cell culturemedia disclosed herein. In some embodiments, said one or morerecombinant polypeptides has reduced amounts of high molecular weightspecies. In some embodiments, said one or more recombinant polypeptideshave reduced amounts of low molecular weight species. In someembodiments, said low molecular weight species comprise polypeptidefragments that are not completely assembled and/or folded. In someembodiments, said high molecular weight species comprise more than onesubunit of a recombinant polypeptide. In some embodiments of any of theembodiments disclosed herein, the percent specific ratio of lowmolecular weight species to the sum of all (1) non-aggregated; (2) lowmolecular weight species; and (3) high molecular weight species ismodulated (e.g. decreased) relative to the percent specific ratiocompared to recombinant polypeptides produced by mammalian cells thatare not cultured in any of the cell culture media disclosed herein. Insome embodiments of any of the embodiments disclosed herein, the percentspecific ratio of high molecular weight species to the sum of all (1)non-aggregated; (2) low molecular weight species; and (3) high molecularweight species is modulated (e.g. decreased) relative to the percentspecific ratio compared to recombinant polypeptides produced bymammalian cells that are not cultured in any of the cell culture mediadisclosed herein. In some embodiments of any of the embodimentsdisclosed herein, said one or more recombinant polypeptides is anantibody or fragment thereof. In some embodiments, said antibody is amonoclonal antibody. In some embodiments, the monoclonal antibodyinhibits the growth of a proliferating cell. In some embodiments, saidantibody or fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin,CD20, CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV),glycoprotein IIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cellactivating factor (BAFF), CD19, CD30, interleukin-1 beta (IL1(3),prostate specific membrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7(CD319), proprotein convertase subtilisin/kexin type 9 (PCSK9),dabigatran, cytotoxic T-lymphocyte-associated protein 4 (CTLA4),interleukin-5 (IL-5), programmed cell death protein (PD-1), VEGFR2(KDR), protective antigen (PA) of B. anthracis, interleukin-17 (IL-17),interleukin-6 (IL-6), interleukin-6 receptor (IL6R), interleukin-12(IL-12), interleukin 23 (IL-23), sclerostin (SOST), myostatin (GDF-8),activin receptor-like kinase 1, delta like ligand 4 (DLL4), angiopoietin3, VEGFR1, selectin, oxidized low-density lipoprotein (oxLDL),platelet-derived growth factor receptor beta, neuropilin 1, VonWillebrand factor (vWF), integrin α_(V)β₃, neural apoptosis-regulatedproteinase 1, integrin α_(IIb)β₃, beta-amyloid, reticulon 4(RTN4)/Neurite Outgrowth Inhibitor (NOGO-A), nerve growth factor (NGF),LINGO-1, myelin-associated glycoprotein, or integrin α4β7. In someembodiments, said monoclonal antibody is trastuzumab, pertuzumab,infliximab, adalimumab, bevacizumab, ranibizumab, natalizumab,rituximab, alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab,cetuximab, panitumumab, palivizumab, abciximab, basiliximab,ibritumomab, omalizumab, eculizumab, abciximab, alirocumab, basiliximab,belimumab, blinatumomab, brentuximab, canakinumab, capromab,daratumumab, denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab,idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab,obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab,raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab,alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab,ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab,inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab,bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab,ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab,gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab,ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab.

In further aspects, provided herein are methods for modulating (e.g.reducing) the amount of acidic or basic charge species of one or morerecombinant polypeptides produced by an engineered mammalian cell, themethod comprising: (a) culturing said mammalian cell in any of the cellculture media disclosed herein under suitable conditions for theproduction of said one or more recombinant polypeptides; and (b)producing said one or more recombinant polypeptides, wherein said one ormore recombinant polypeptides have reduced amounts of acidic chargespecies compared to recombinant polypeptides produced by mammalian cellsthat are not cultured in any of the cell culture media disclosed herein.In some embodiments, the percent specific ratio of acidic or basiccharge species to the total sum of all (1) acidic species; (2) mainspecies; and (3) basic charge species is reduced relative to recombinantpolypeptides produced by mammalian cells that are not cultured in any ofthe cell culture media disclosed herein. In some embodiments of any ofthe embodiments disclosed herein, said one or more recombinantpolypeptides is an antibody or fragment thereof. In some embodiments ofany of the embodiments disclosed herein, said antibody is a monoclonalantibody. In some embodiments, said antibody or fragment thereof bindsto HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR,respiratory syncytial virus (RSV), glycoprotein IIb/IIIa, IgG1, IgE,complement component 5 (C5), B-cell activating factor (BAFF), CD19,CD30, interleukin-1 beta (IL113), prostate specific membrane antigen(PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertasesubtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7. In some embodiments of any of the embodiments disclosedherein, said monoclonal antibody is trastuzumab, pertuzumab, infliximab,adalimumab, bevacizumab, ranibizumab, natalizumab, rituximab,alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab, cetuximab,panitumumab, palivizumab, abciximab, basiliximab, ibritumomab,omalizumab, eculizumab, abciximab, alirocumab, basiliximab, belimumab,blinatumomab, brentuximab, canakinumab, capromab, daratumumab,denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab,idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab,obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab,raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab,alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab,ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab,inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab,bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab,ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab,gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab,ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab. In someembodiments of any of the embodiments disclosed herein, the medium is abasal medium. In some embodiments of any of the embodiments disclosedherein, the medium is a feed medium.

In another aspect, provided herein is a kit comprising: (a) (i) amammalian cell culture basal medium and/or (ii) a mammalian cell culturefeed medium; and (b) one or more sources of lithium ions. In someembodiments of any of the embodiments disclosed herein, said kit furthercomprises (c) ethanol and/or (d) one or more fatty acids. In someembodiments, said one or more fatty acids is selected from Butyric (C4),Valeric (C5), Caproic (C6), Enanthic (C7), Caprylic (C8), Pelargonic(C9), Capric (C10), Undecylic (C11), Lauric (C12), Tridecylic (C13),Myristic (C14), Pentadecanoic (C15), Palmitic (C16), Margaric (C17),Stearic (C18), Nonadecylic (C19), Arachidic (C20), Heneicosylic (C21),Behenic (C22), Tricosylic (C23), Lignoceric (C24), Pentacosylic (C25),Cerotic (C26), Heptacosylic (C27), Montanic (C28), Nonacosylic (C29),Melissic (C30), Hentriacontylic (C31), Lacceroic (C32), Psyllic (C33),Geddic (C34), Ceroplastic (C35), Hexatriacontylic (C36),Heptatriacontanoic (C37), or Octatriacontanoic (C38) acids. In someembodiments of any of the embodiments disclosed herein, said kit furthercomprises (e) written instructions for culturing mammalian cells. Insome embodiments of any of the embodiments disclosed herein, said one ormore sources of lithium ions is selected from the group of one or moreof lithium acetate, lithium chloride, lithium carbonate, lithiumoxybutyrate, lithium orotate, lithium bromide, lithium citrate, lithiumfluoride, lithium iodide, lithium nitrate, and lithium sulfate.

In other aspects, provided herein are recombinant polypeptides producedby culturing an engineered mammalian cell in any of the cell culturemedia disclosed herein under suitable conditions for the production ofsaid recombinant polypeptide. In some embodiments, said polypeptide isan antibody or fragment thereof. In some embodiments of any of theembodiments disclosed herein, said antibody is a monoclonal antibody. Insome embodiments, the monoclonal antibody inhibits the growth of aproliferating cell. In some embodiments, said antibody or fragmentthereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25,CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa,IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF),CD19, CD30, interleukin-1 beta (IL10), prostate specific membraneantigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertasesubtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₂,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7. In some embodiments, said monoclonal antibody istrastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab,ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab,efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of major N-glycan linkedspecies following the typical maturation pathway. Glycosylationprocessing begins in the Endoplasmic Reticulum with further processingin the cis-, medial- and trans-Golgi. Major glycan species shown,include: mannose-6-N-acetylglycosamine-2 (Man6),mannose-5-N-acetylglycosamine-2 (Man5), mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F),mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose-1-N-acetylneuraminic-1(G1F-NANA) andmannose-3-N-acetylglucosamine-4-galactose-2-fucose-1-N-acetylneuraminic-2(G2F-2NANA).

FIG. 2 depicts impact on titer and Man5% glycan species. Titer isreported as volumetric titer and Man5% glycan species as percent of thetotal detected glycan pool. Values are normalized to baseline. Lithiumwas fed daily to increase bioreactor concentration by 0.11 mM (lowlithium) and 1.11 mM (high lithium) in a 12 day fed-batch process.Higher doses of lithium increased titer over 21% and reduced Man5% byover 13% compared to baseline

FIG. 3 depicts the prediction response profile for Titer, Man5%, G0%,HMW %, LMW %, Acidic % and Basic % species using 2 L bench scalebioreactors. Lithium was fed daily to increase bioreactor concentrationsby 1 mM in a 14 day fed-batch process. Values are normalized to highestobserved value for each response. Dotted lines represent confidenceintervals of each response.

FIG. 4 depicts the prediction response profile for Titer, Man5%, G0%,G0F %, G1F %, G2F %, HMW %, LMW %, Acidic % and Basic % species.Physical conditions were altered by changing temperature and pH setpoints. Lithium was fed daily to increase bioreactor workingconcentration by 0.5 mM or 1 mM in a 12 day fed-batch process. Responsevalues are normalized to the highest observed value for each response.Dotted lines represent confidence intervals of each response.

FIG. 5 depicts the combined prediction response profile of Titer, Man5%,G0%, G0F %, G1F %, G2F %, HMW %, LMW %, Acidic % and Basic % species.Lithium was fed daily to increase bioreactor working concentrations by0.5 mM or 1 mM in a 12 day fed-batch process. Response values arenormalized to the highest observed value for each response. Dotted linesrepresent confidence intervals of each response.

FIG. 6 depicts impact on Man6%, Man5% and HMW % levels using a CHO DG44host producing mAb2. Man6% and Man5% glycan species are reported as apercent of the total detected glycan pool. HMW % is reported as apercent of the total detected polypeptide molecular weight variants.Values are normalized to baseline. Lithium was fed daily to increasebioreactor concentrations by 0.11 mM (Low Lithium), 0.44 mM (MediumLithium) and 1.11 mM (High Lithium) in a 13 day fed-batch process. Highlithium reduced Man6% and Man5% glycan species by 29% and 12%,respectively over baseline. High lithium reduced high molecular weightspecies by 24% compared to baseline.

FIG. 7 depicts the impact on Man5%, G0%, G0F %, G1F %, G2F %, Acidic %and Basic % species using a CHO.DXB11 host (Cell Line A) producing mAb1.Man5%, G0%, G0F %, G1F % and G2F % glycan species are reported as apercent of the total detected glycan pool. Acidic % and Basic % arereported as a percent of the total detected polypeptide charge speciesvariants. Values are normalized to baseline. Ethanol was fed daily toincrease bioreactor concentrations by 0.0% (Baseline) and 0.057% (withEthanol) v/v in a 12 day fed-batch process.

FIG. 8 depicts the impact on Man5%, G0%, G0F %, G1F %, G2F %, Acidic %and Basic % species using a CHO.DXB11 host (Cell Line B) producing mAb1.Man5%, G0%, G0F %, G1F % and G2F % glycan species are reported as apercent of the total detected glycan pool. Acidic % and Basic % arereported as a percent of the total detected polypeptide charge speciesvariants. Values are normalized to baseline. Ethanol was fed daily toincrease bioreactor concentrations by 0.0% (Baseline) and 0.057% (withEthanol) v/v in a 12 day fed-batch process.

FIG. 9 depicts the prediction response profile of Titer, Man5%, G0%,Acidic % and Basic % species. Ethanol was fed daily to increasebioreactor concentrations by 0.0% or 0.1% v/v in a 12 day fed-batchprocess. Response values are normalized to the highest observed valuefor each response. Dotted lines represent confidence intervals of eachresponse.

FIG. 10 depicts the impact on Man5%, G0%, G0F %, G1F %, G2F %, HMW % andLMW % species using bench-scale bioreactors. Man5%, G0%, G0F %, G1F %and G2F % glycan species are reported as a percent of the total detectedglycan pool. HMW % and LMW % are reported as a percent of the totaldetected polypeptide molecular weight variants. Values are normalized tolow ethanol. Ethanol was fed daily to increase bioreactor concentrationsby 0.072% (Low Ethanol) and 0.144% (High Ethanol) v/v in a 14 dayfed-batch process.

FIG. 11 depicts the impact on Man5%, G0%, G0F %, G1F %, G2F %, Acidic %and Basic % species using two different feed formulations. The samebasal media was used in formulation 1 and 2. Formulation 1 and 2 feedswere very similar in composition, containing the same type and amount ofamino acids and vitamins. Salt and metal ion type and concentration wereslightly varied. Man5%, G0%, G0F %, G1F % and G2F % glycan species arereported as a percent of the total detected glycan pool. Acidic % andBasic % are reported as a percent of the total detected polypeptidecharge species variants. Values are normalized to Baseline lackingethanol. Ethanol was fed daily to increase bioreactor concentrations by0.0% (Baseline) and 0.018% (Formulation 1 and Formulation 2) v/v in a 14day fed-batch process.

FIG. 12 depicts the impact on Man5%, G0%, G0F %, G1F %, G2F % and HMW %using microscale bioreactors and a CHO.DG44 cell line to produce mAb 2.Man5%, G0%, G0F %, G1F % and G2F % glycan species are reported as apercent of the total detected glycan pool. HMW % is reported as apercent of the total detected polypeptide molecular weight variants.Values are normalized to Baseline. Ethanol was fed daily to increasebioreactor concentrations by 0.0% (Baseline), 0.05% (Low Ethanol) and0.15% (High Ethanol) v/v in a 13 day fed-batch process.

FIG. 13 depicts the synergistic impact on Titer, Man5%, G0%, G0F %, G1F%, G2F %, HMW %, LMW %, Acidic % and Basic % using bench-scalebioreactors supplemented with both ethanol and oleic acid. Titer isreported as volumetric titer. Man5%, G0%, G0F %, G1F % and G2F % glycanspecies are reported as a percent of the total detected glycan pool. HMW% and LMW % are reported as a percent of the total detected polypeptidemolecular weight variants. Acidic % and Basic % are reported as apercent of the total detected polypeptide charge species variants.Values are normalized to Baseline, which contains no ethanol or oleicacid. Ethanol was fed daily to increase bioreactor concentrations by0.0% (Baseline) and 0.057% (w/Ethanol+Oleic Acid) v/v and oleic acid wasfed daily to increase bioreactor concentrations by 0 μM (Baseline) and40 μM (w/Ethanol+Oleic Acid) in a 14 day fed-batch process.

FIG. 14 depicts the impact on Titer, Man5%, G0%, G0F %, G1F %, G2F %,HMW %, LMW %, Acidic % and Basic % using bench-scale bioreactors. Titeris reported as volumetric titer. Man5%, G0%, G0F %, G1F % and G2F %glycan species are reported as a percent of the total detected glycanpool. HMW % and LMW % are reported as a percent of the total detectedpolypeptide molecular weight variants. Acidic % and Basic % are reportedas a percent of the total detected polypeptide charge species variants.Values are normalized to Baseline w/Ethanol. Oleic acid was fed daily toincrease bioreactor concentrations by 0 μM (Baseline w/Ethanol) and 40μM (w/Ethanol+Oleic Acid) in a 14 day fed-batch process.

FIG. 15A depicts the impact of spiking cell culture medium with sodiumchloride on Titer, Man5%, G0%, G0F %, G1F, G2F %, Acidic % and Basic %species using microscale bioreactors. Process strategy 1 involved asingle temperature shift strategy. Titer is reported as volumetrictiter. Man5%, G0%, G0F %, G1F % and G2F % glycan species are reported asa percent of the total detected glycan pool. Acidic % and Basic % arereported as a percent of the total detected polypeptide charge speciesvariants. Values are normalized to baseline. Sodium chloride was feddaily to increase bioreactor concentrations by 4.59 mM (L NaCl) and 9.18mM (H NaCl) in a 12 day fed-batch process.

FIG. 15B depicts the impact of spiking cell culture medium with sodiumchloride on Titer, Man5%, G0%, G0F %, G1F, G2F %, Acidic % and Basic %species using microscale bioreactors. Process strategy 2 involved a dualtemperature shift strategy. Titer is reported as volumetric titer.Man5%, G0%, G0F %, G1F % and G2F % glycan species are reported as apercent of the total detected glycan pool. Acidic % and Basic % arereported as a percent of the total detected polypeptide charge speciesvariants. Values are normalized to baseline. Sodium chloride was feddaily to increase bioreactor concentrations by 4.59 mM (L NaCl) and 9.18mM (H NaCl) in a 12 day fed-batch process.

FIG. 16 depicts the impact of addition of various types of fatty acidsto the culture medium with respect to titer for two separate monoclonalantibodies (mAb1 and mAb4) produced by two separate cell lines. Titer isreported as volumetric titer. Values are normalized to control.

FIG. 17A depicts the impact of addition of various types of fatty acidsto the culture medium with respect to high molecular weight (BMW)species for two separate monoclonal antibodies (mAb1 and mAb4) producedby two separate cell lines. BMW % is reported as a percent of the totaldetected polypeptide molecular weight variants. Values are normalized tocontrol.

FIG. 17B depicts the impact of addition of various types of fatty acidsto the culture medium with respect to low molecular weight (LMW) speciesfor two separate monoclonal antibodies (mAb1 and mAb4) produced by twoseparate cell lines. LMW % is reported as a percent of the totaldetected polypeptide molecular weight variants. Values are normalized tocontrol.

FIG. 18A depicts the impact of addition of various types of fatty acidsto the culture medium with respect to acidic charge species for twoseparate monoclonal antibodies (mAb1 and mAb4) produced by two separatecell lines. Acidic % is reported as a percent of the total detectedpolypeptide charge species variants. Values are normalized to control.

FIG. 18B depicts the impact of addition of various types of fatty acidsto the culture medium with respect to basic charge species for twoseparate monoclonal antibodies (mAb1 and mAb4) produced by two separatecell lines. Basic % is reported as a percent of the total detectedpolypeptide charge species variants. Values are normalized to control.

FIG. 19A depicts the impact of addition of various types of fatty acidsto the culture medium with respect to Man5% glycan species for twoseparate monoclonal antibodies (mAb1 and mAb4) produced by two separatecell lines. Man5% glycan species is reported as a percent of the totaldetected glycan pool. Values are normalized to control.

FIG. 19B depicts the impact of addition of various types of fatty acidsto the culture medium with respect to G0% glycan species for twoseparate monoclonal antibodies (mAb1 and mAb4) produced by two separatecell lines. G0% glycan species is reported as a percent of the totaldetected glycan pool. Values are normalized to control.

FIG. 20 depicts the impact of addition of various types of fatty acidsto the culture medium with respect to G0F % glycan species for twoseparate monoclonal antibodies (mAb1 and mAb4) produced by two separatecell lines. G0F % glycan species is reported as a percent of the totaldetected glycan pool. Values are normalized to control.

DETAILED DESCRIPTION

This invention provides, inter alia, methods, compositions, and kits forthe culturing of mammalian cells. The invention is based, in part, onthe inventors' discovery that culturing mammalian cells geneticallyengineered to produce one or more recombinant polypeptides in a mediumcontaining one or more of a lithium ion source, ethanol, and/or one ormore fatty acids resulted in increased polypeptide titer as well asreduced high and low molecular weight species. Additionally, use of thecompositions described herein resulted in polypeptide products having amore favorable glycosylation profile compared to mammalian cells thatare not cultured in the media described herein. Consequently, use of thecell culture media compositions disclosed herein can not only increasethe amount of product produced by engineered mammalian cells, therebyresulting in more favorable production economics, but can also result inproducts with glycosylation profiles that impart added benefits for invivo administration of those products, such as improved pharmacokinetics(PK) and/or pharmacodynamics (PD).

I. GENERAL TECHNIQUES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology,which are well known to those skilled in the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, fourth edition (Sambrook et al., 2012) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2014); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Antibodies: A Laboratory Manual, Second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(Greenfield, ed., 2014), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000,(including supplements through 2014), Gene Transfer and Expression inMammalian Cells (Makrides, ed., Elsevier Sciences B.V., Amsterdam,2003), and Current Protocols in Immunology (Horgan K and S. Shaw (1994)(including supplements through 2014).

II. DEFINITIONS

A “basal medium,” as used herein, is a medium used for culturingeukaryotic cells which is, itself, directly used to culture the cellsand is not used as an additive to other media, although variouscomponents may be added to a basal medium. For example, if CHO cellswere cultured in DMEM, a well-known, commercially-available medium formammalian cells, and periodically fed with glucose or other nutrients,DMEM would be considered the basal medium. Other examples of basal mediainclude, without limitation, MEM medium, IMDM medium, 199/109 medium,HamF10/F12 medium, McCoy's 5A medium, and RPMI 1640 medium.

A “feed medium” is a medium used as a feed in a culture of eukaryoticcells, which may be, for example, mammalian cells. A feed medium, like abasal medium, is designed with regard to the needs of the particularcells being cultured. Thus, a basal medium can be used as a basis fordesigning a feed medium. As described below in more detail, a feedmedium can have higher concentrations of most, but not all, componentsof a base culture medium. For example, some components, such as, forexample, nutrients including amino acids or carbohydrates, may be atabout 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×,100×, 200×, 400×, 600×, 800×, or even about 1000× of their normalconcentrations in a basal medium. Other components, such as salts, maybekept at about 1× of the basal medium concentration, as one would want tokeep the feed isotonic with the basal medium. Thus, in some embodiments,various components are added to keep the feed medium physiologic andothers are added because they replenish nutrients to the cell culture.

A “recombinant polypeptide” or a “recombinant protein” is a proteinresulting from the process of genetic engineering. Cells are“genetically engineered” when recombinant nucleic acid sequences thatallow expression of the recombinant protein have been introduced intothe cells such as viral infection with a recombinant virus,transfection, transformation, or electroporation. See e.g. Kaufman etal. (1990), Meth. Enzymol. 185: 487-511; Current Protocols in MolecularBiology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, andquarterly updates through 2015). In some embodiments, a recombinantpolypeptide is an antibody or functional fragment thereof.

The term “genetic engineering” refers to a recombinant DNA or RNA methodused to create a host cell that expresses a gene at elevated levels orat lowered levels, or expresses a mutant form of the gene. In otherwords, the cell has been transfected, transformed or transduced with arecombinant polynucleotide molecule, and thereby altered so as to causethe cell to alter expression of a desired protein. The methods of“genetic engineering” also encompass numerous methods including, but notlimited to, amplifying nucleic acids using polymerase chain reaction,assembling recombinant DNA molecules by cloning them in Escherichiacoli, restriction enzyme digestion of nucleic acids, ligation of nucleicacids, and transfer of bases to the ends of nucleic acids, amongnumerous other methods that are well-known in the art. See e.g. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ColdSpring Harbor Laboratory, 1989, as well as updates through the present

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

III. COMPOSITIONS OF THE INVENTION

In some aspect, provided herein is a medium for culturing mammaliancells containing a basal medium or a feed medium as well as one or moreof (1) a source of lithium ions, (2) ethanol, and/or (3) one or morefatty acids.

A. Mammalian Cells

Any mammalian cell capable of being cultured is suitable for use in thepresent invention. This includes cells derived from humans (for example,HeLa cells) as well as cells derived from rodents such as mice, rats,hamsters (for example Chinese hamster ovary (CHO) cells), or Guineapigs. It also includes mammalian cells derived from other species,particularly primate species.

Examples of mammalian cells that can be cultured in the media accordingto the present invention include, without limitation, murine C127 cells,3T3 cells, COS cells, human osteosarcoma cells, MRC-5 cells, babyhamster kidney (BHK) cells, VERO cells, HEK 293 cells, rHEK 293 cells,normal human fibroblast cells, Stroma cells, Hepatocytes cells, orPER.C6 cells. Examples of hybridomas that may be cultured in the processaccording to the present invention include, e.g., DA4.4 calls, 123Acells, 127A cells, GAMMA cells and 67-9-B cells.

Further examples of mammalian cells appropriate for use with thecompositions disclosed herein are COP cells, L cells, C 127 cells, Sp2/0cells, NS-O cells, NS-I cells, NIH3T3 cells, PC12 cells, PC12h cells,COS1 cells, COS3 cells, COST cells, CV1 cells, Chinese hamster ovary(CHO) cells, African green monkey kidney (AGMK) cells, or a cell derivedfrom diploid fibroblasts, from cancer cells (such as myeloma cells), andHepG2 cells.

B. Cells Genetically Engineered to Produce Recombinant Polypeptides

In another aspect, the mammalian cells cultured in any of the mediadescribed herein can be genetically engineered to produce one or morerecombinant polypeptides. For purposes of the present invention,mammalian cells can be genetically engineered in accordance with anymethod known in the art. For example, expression vectors can be designedto contain nucleic acid sequences which optimize gene expression forcertain host mammalian cell strains. Such optimization componentsinclude, but are not limited to origin of replication, promoters, andenhancers. The vectors and components referenced herein are describedfor exemplary purposes and are not meant to narrow the scope of theinvention. Suitable vectors are those which are compatible with themammalian host cell employed. Suitable vectors can be derived, forexample, from a bacterium, a virus (such as bacteriophage T7 or an M-13derived phage), a cosmid, a yeast, or a plant. Suitable vectors can bemaintained in low, medium, or high copy number in the mammalian hostcell. Protocols for obtaining and using such vectors are known to thosein the art (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor, 2012).

A nucleic acid sequence encoding a desired polypeptide or an expressionvector comprising the same can be inserted into a mammalian cell (e.g.,a CHO cell or any other mammalian cell described herein) using standardtechniques for introduction of a DNA construct or vector into amammalian host cell, such as transformation, electroporation, nuclearmicroinjection, transduction, transfection (e.g., lipofection mediatedor DEAE-Dextrin mediated transfection or transfection using arecombinant phage virus), incubation with calcium phosphate DNAprecipitate, high velocity bombardment with DNA-coated microprojectiles,and protoplast fusion. General transformation techniques are known inthe art (see, e.g., Molecular Cloning: A Laboratory Manual, 4th ed.,Cold Spring Harbor, 2012). The introduced nucleic acids may beintegrated into chromosomal DNA or maintained as extrachromosomalreplicating sequences. Transformants can be selected by any method knownin the art.

C. Sources of Lithium Ions

In some aspects of any of the mammalian cell culture media describedherein, the medium contains one or more sources of lithium ions. Anylithium ion source is suitable for use in the present invention andincludes lithium salts, solvates, and metal.

Nonlimiting sources of lithium ions include lithium hydroxide (LiOH andLiOH.H₂O), lithium carbonate (Li₂CO₃), lithium methoxide, lithiumacetate, lithium oxide (Li₂O) lithium peroxide (Li₂O₂), lithium hydride(LiH), lithium chloride (LiCl), lithium fluoride (LiF), lithium iodide(LiI), lithium sulfide (Li₂S), lithium sulfite (LiSO₃), lithium sulfate(LiSO₄), lithium superoxide (LiO₂), lithium carbide (Li₂C₂), lithiumtetrachloroaluminate (LiAlCl₄), lithium aluminum hydride (LiAlH₄),lithium aluminium oxide (LiAlO₂), lithium tetrafluoroborate (LiBF₄),lithium borohydride (LiBH₄), lithium metaborate (LiBO₂), lithiumtetraborate (Li₂B₄O₇), lithium triborate (LiB₃O₅), lithium hypochlorite(LiClO), lithium chlorate (LiClO₃), lithium perchlorate (LiClO₄),lithium cobalt oxide (LiCoO₂), lithium iodate (LiIO₃), lithium amide(LiNH₂), lithium imide (Li₂NH), liithium azide (LiN₃), lithium nitrite(LiNO₂), lithium nitrate (LiNO₃), lithium tetraborate (Li₂B₄O₇), lithiumcarbide (Li₂C₂), and lithium molybdate (Li₂MoO₄).

In some embodiments, the amount of the lithium ion source present in themedium (for example, a basal medium or a feed medium) can range fromabout 0.05 mM to about 15 mM, such as any of about 0.05 mM to about 0.5mM, about 0.1 mM to about 0.75 mM, about 0.25 mM to about 1 mM, about0.5 mM to about 1.25 mM, about 0.75 mM to about 1.5 mM, about 1 mM toabout 1.75 mM, about 1.25 mM to about 1.75 mM, about 1.25 mM to about2.25 mM, about 1.5 mM to about 2.5 mM, about 1.75 mM to about 2.75 mM,about 2 mM to about 3 mM, about 2.5 mM to about 3.5 mM, about 3 mM toabout 4 mM, about 3.5 mM to about 4.5 mM, about 4 mM to about 5 mM,about 4.5 mM to about 5.5 mM, about 5 mM to about 6 mM, about 5.5 mM toabout 6.5 mM, about 6 mM to about 7 mM, about 6.5 mM to about 8.5 mM,about 7.5 mM to about 9.5 mM, about 8.5 mM to about 11.5 mM, about 9.5mM to about 12.5 mM, about 11.5 mM to about 13.5 mM, about 12.5 mM toabout 15 mM, about 14 mM to about 17 mM, about 16 mM to about 19 mM,about 18 mM to about 21 mM, about 20 mM to about 23 mM, about 22 mM toabout 25 mM, about 0.75 mM to about 1.75 mM, about 0.5 mM to about 10mM, about 0.5 mM to about 7.5 mM, about 1 mM to about 5 mM, about 1 mMto about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, orabout 1 mM to about 1.25 mM.

In other embodiments, the amount of the lithium ion source present inthe medium (for example, a basal medium or a feed medium) is any ofabout 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4mM, 0.45 mM, 0.5 mM, 0.55 mM, 0.6 mM, 0.65 mM, 0.7 mM, 0.75 mM, 0.8 mM,0.85 mM, 0.9 mM, 0.95 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM,1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM,2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM,3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM,4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM, 5.1 mM,5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM,6.1 mM, 6.2 mM, 6.3 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM,7 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM,7.9 mM, 8 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6 mM, 8.7 mM,8.8 mM, 8.9 mM, 9 mM, 9.1 mM, 9.2 mM, 9.3 mM, 9.4 mM, 9.5 mM, 9.6 mM,9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5mM, 10.6 mM, 10.7 mM, 10.8 mM, 10.9 mM, 11 mM, 11.1 mM, 11.2 mM, 11.3mM, 11.4 mM, 11.5 mM, 11.6 mM, 11.7 mM, 11.8 mM, 11.9 mM, 12 mM, 12.1mM, 12.2 mM, 12.3 mM, 12.4 mM, 12.5 mM, 12.6 mM, 12.7 mM, 12.8 mM, 12.9mM, 13 mM, 13.1 mM, 13.2 mM, 13.3 mM, 13.4 mM, 13.5 mM, 13.6 mM, 13.7mM, 13.8 mM, 13.9 mM, 14 mM, 14.1 mM, 14.2 mM, 14.3 mM, 14.4 mM, 14.5mM, 14.6 mM, 14.7 mM, 14.8 mM, 14.9 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, or more, inclusive of allvalues falling in between these numbers.

D. Fatty Acids

In some aspects of any of the mammalian cell culture media describedherein, the medium contains one or more fatty acids. Any fatty acid issuitable for use in the present invention. As used herein, “fatty acid”refers to any member of the family of naturally occurring orsynthetically produced hydrocarbons containing a carboxylic acid, and atleast one saturated, unsaturated, partially unsaturated, or conjugatedcarbon-carbon bond. Furthermore, the term fatty acid is used generallyto describe fatty acids, C2-C38 fatty acids, conjugated fatty acids,lipids, phospholipids, oils, fats, triacylglycerides, fatty acidderivatives, diacyl glycerol, isoprenoids, sphingolipids, glycerolipids,and the like.

In some embodiments, the fatty acid is a saturated fatty acid such as,without limitation, one or more of Butyric (C4), Valeric (C5), Caproic(C6), Enanthic (C7), Caprylic (C8), Pelargonic (C9), Capric (C10),Undecylic (C11), Lauric (C12), Tridecylic (C13), Myristic (C14),Pentadecanoic (C15), Palmitic (C16), Margaric (C17), Stearic (C18),Nonadecylic (C19), Arachidic (C20), Heneicosylic (C21), Behenic (C22),Tricosylic (C23), Lignoceric (C24), Pentacosylic (C25), Cerotic (C26),Heptacosylic (C27), Montanic (C28), Nonacosylic (C29), Melissic (C30),Hentriacontylic (C31), Lacceroic (C32), Psyllic (C33), Geddic (C34),Ceroplastic (C35), Hexatriacontylic (C36), Heptatriacontanoic (C37), orOctatriacontanoic (C38) acids.

In other embodiments, the fatty acid is an omega-3 (ω-3) unsaturatedfatty acid such as, without limitation, one or more of α-Linolenic(18:3), Stearidonic (18:4), Eicosapentaenoic (20:5), or Docosahexaenoic(22:6) acids.

In additional embodiments, the fatty acid is an omega-6 (ω-6)unsaturated fatty acid such as, without limitation, one or more ofLinoleic (18:2), γ-Linolenic (18:3), Dihomo-γ-linolenic (20:3),Arachidonic (20:4), or Adrenic (22:4) acids.

In yet other embodiments, the fatty acid is an omega-7 (ω-7) unsaturatedfatty acid such as, without limitation, one or more of Palmitoleic(16:1), Vaccenic (18:1), or Paullinic (20:1) acids.

In another embodiment, the fatty acid is an omega-9 (ω-9) unsaturatedfatty acid such as, without limitation, one or more of Oleic (18:1),Elaidic (trans-18:1), Gondoic (20:1), Erucic (22:1), Nervonic (24:1), orMead (20:3) acid.

As used herein, the term “isoprenoid” refers to a large and diverseclass of naturally-occurring class of organic compounds composed of twoor more units of hydrocarbons, with each unit consisting of five carbonatoms arranged in a specific pattern. As used herein, the term“terpenoid” refers to a large and diverse class of organic moleculesderived from five-carbon isoprenoid units assembled and modified in avariety of ways and classified in groups based on the number ofisoprenoid units used in group members. Hemiterpenoids have oneisoprenoid unit. Monoterpenoids have two isoprenoid units.Sesquiterpenoids have three isoprenoid units. Diterpenoids have fourisoprene units. Sesterterpenoids have five isoprenoid units.Triterpenoids have six isoprenoid units. Tetraterpenoids have eightisoprenoid units. Polyterpenoids have more than eight isoprenoid units.

In some embodiments, the amount of fatty acids present in the medium(for example, a basal medium or a feed medium) can range from about 1 μMto about 1 mM, such as any of about 1 μM to about 5 μM, about 1 μM toabout 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about1 μM to about 25 μM, about 1 μM to about 30 μM, about 1 μM to about 35μM, about 1 μM to about 40 μM, about 1 μM to about 45 μM, about 1 μM toabout 50 μM, about 1 μM to about 55 μM, about 1 μM to about 60 μM, about1 μM to about 65 μM, about 1 μM to about 70 μM, about 1 μM to about 75μM, about 1 μM to about 80 μM, about 10 μM to about 20 μM, about 10 μMto about 30 μM, about 10 μM to about 40 μM, about 10 μM to about 50 μM,about 10 μM to about 60 μM, about 10 μM to about 70 μM, about 10 μM toabout 80 μM, about 10 μM to about 90 μM, about 20 μM to about 40 μM,about 20 μM to about 60 μM, about 20 μM to about 80 μM, about 20 μM toabout 100 μM, about 30 μM to about 50 μM, about 30 μM to about 60 μM,about 30 μM to about 70 μM, about 30 μM to about 80 μM, about 30 μM toabout 90 μM, about 40 μM to about 50 μM, about 40 μM to about 60 μM,about 40 μM to about 80 μM, about 40 μM to about 90 μM, about 50 μM toabout 60 μM, about 50 μM to about 70 μM, about 50 μM to about 80 μM,about 50 μM to about 90 μM, about 50 μM to about 100 μM, about 60 μM toabout 70 μM, about 60 μM to about 80 μM, about 60 μM to about 90 μM,about 60 μM to about 100 μM, about 70 μM to about 80 μM, about 70 μM toabout 90 μM, about 70 μM to about 100 μM, about 50 μM to about 150 μM,about 100 μM to about 200 μM, about 150 μM to about 250 μM, about 200 μMto about 300 μM, about 250 μM to about 350 μM, about 300 μM to about 400μM, about 350 μM to about 450 μM, about 400 μM to about 500 μM, about450 μM to about 550 μM, about 500 μM to about 600 μM, about 550 μM toabout 650 μM, about 600 μM to about 700 μM, about 650 μM to about 750μM, about 700 μM to about 800 μM, about 750 μM to about 850 μM, about800 μM to about 900 μM, about 850 μM to about 950 μM, about or 900 μM toabout 1 mM.

In other embodiments, the amount of the fatty acids present in themedium (for example, a basal medium or a feed medium) is any of about 1μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM,13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63μM, 64 μM, 65 μM, 66 μM, 67 μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73μM, 74 μM, 75 μM, 76 μM, 77 μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83μM, 84 μM, 85 μM, 86 μM, 87 μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93μM, 94 μM, 95 μM, 96 μM, 97 μM, 98 μM, 99 μM, 100 μM, 125 μM, 150 μM,175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM,400 μM, 425 μM, 450 μM, 475 μM, 500 μM, 525 μM, 550 μM, 575 μM, 600 μM,625 μM, 650 μM, 675 μM, 700 μM, 725 μM, 750 μM, 775 μM, 800 μM, 825 μM,850 μM, 875 μM, 900 μM, 925 μM, 950 μM, 975 μM, or 1 mM, inclusive ofall numbers falling in between these values.

In some embodiments, the fatty acid is one or more of thymol,cholesteryl acetate, methyl octanoate, 1-octanoyl-rac-glycerol, oleicacid, linoleic acid, linolenic acid, cholesterol, palmitic acid, stearicacid, and myristic acid.

In one embodiment, cell culture media supplementation with one or moreof thymol, 1-octanoyl-rac-glycerol, linoleic acid, and/or linolenic acidincreases the titer of recombinant protein produced by cell lines grownin the media by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive ofpercentages falling in between these values, relative to the titer ofthe recombinant protein produced in media not supplemented with one ormore of the fatty acids.

In some embodiments, the phrase “modulating” or “modulates” with respectto the relative percentage of 1) a HMW and/or LMW species; or 2) anacidic and/or basic charge species refers to increasing the relativepercentage of 1) a particular HMW and/or LMW species; or 2) a particularacidic and/or basic charge species of a recombinant polypeptide.However, in other embodiments, the phrase “modulating” or “modulates”with respect to the relative percentage of 1) a HMW and/or LMW species;or 2) an acidic and/or basic charge species refers to decreasing therelative percentage of 1) a particular HMW and/or LMW species; or 2) aparticular acidic and/or basic charge species of a recombinantpolypeptide.

In another embodiment, cell culture media supplementation with one ormore of thymol, cholesterol acetate, methyl octanoate,1-octanoyl-rac-glycerol, palmitic acid, stearic acid, and myristic acidmodulates the relative percentage of HMW species produced by recombinantprotein-producing cell lines grown in the media by any of about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive of percentages falling in between thesevalues, relative to the amount of HMW species of the recombinant proteinproduced in media not supplemented with one or more of the fatty acids.

In a further embodiment, cell culture media supplementation with one ormore of thymol, cholesteryl acetate, 1-octanoyl-rac-glycerol, linoleicacid, linolenic acid, palmitic acid, and stearic acid modulates therelative percentage of LMW species produced by recombinantprotein-producing cell lines grown in the media by any of about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive of percentages falling in between thesevalues, relative to the amount of LMW species of the recombinant proteinproduced in media not supplemented with one or more of the fatty acids.

In yet other embodiments, cell culture media supplementation with one ormore of thymol, cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid,palmitic acid, and myristic acid modulates the relative percentage ofacidic charge species produced by recombinant protein-producing celllines grown in the media by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive of percentages falling in between these values, relative tothe amount of acidic charge species of the recombinant protein producedin media not supplemented with one or more of the fatty acids.

In additional embodiments, cell culture media supplementation with oneor more of thymol, cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, cholesterol, palmitic acid, stearic acid, andmyristic acid modulates the relative percentage of basic charge speciesproduced by recombinant protein-producing cell lines grown in the mediaby any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive of percentagesfalling in between these values, relative to the amount of basic chargespecies of the recombinant protein produced in media not supplementedwith one or more of the fatty acids.

In another embodiment, cell culture media supplementation with one ormore of thymol, cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid,cholesterol, palmitic acid, stearic acid, and myristic acid modulates(for example, increases or decreases) the relative percentage of Man5%glycan species produced by recombinant protein-producing cell linesgrown in the media by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive of percentages falling in between these values, relative tothe amount of Man5% glycan species of the recombinant protein producedin media not supplemented with one or more of the fatty acids.

In still further embodiments, cell culture media supplementation withone or more of thymol, cholesteryl acetate, methyl octanoate, oleicacid, linoleic acid, linolenic acid, cholesterol, palmitic acid, stearicacid, and myristic acid modulates (for example, increases or decreases)the relative percentage of G0% glycan species produced by recombinantprotein-producing cell lines grown in the media by any of about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive of percentages falling in between thesevalues, relative to the amount of G0% glycan species of the recombinantprotein produced in media not supplemented with one or more of the fattyacids.

In additional embodiments, cell culture media supplementation with oneor more of cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, oleic acid, and myristic acid modulates (forexample, increases or decreases) the relative percentage of G0F % glycanspecies produced by recombinant protein-producing cell lines grown inthe media by any of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive ofpercentages falling in between these values, relative to the amount ofG0F % glycan species of the recombinant protein produced in media notsupplemented with one or more of the fatty acids.

E. Ethanol

In further aspects of any of the mammalian cell culture media describedherein, the medium contains ethanol.

In some embodiments, the amount of ethanol present in the medium (forexample, a basal medium or a feed medium) can range from about 0.001% toabout 4% (v/v), such as any of about 0.001% to about 0.01% (v/v), about0.001% to about 0.02% (v/v), about 0.001% to about 0.05% (v/v), about0.001% to about 0.075% (v/v), about 0.001% to about 0.09% (v/v), about0.001% to about 0.1% (v/v), about 0.001% to about 0.15% (v/v), about0.001% to about 0.2% (v/v), about 0.001% to about 0.25% (v/v), about0.001% to about 0.3% (v/v), about 0.05% to about 0.075% (v/v), about0.05% to about 0.1% (v/v), about 0.05% to about 0.15% (v/v), about 0.05%to about 0.2% (v/v), about 0.05% to about 0.25% (v/v), about 0.05% toabout 0.3% (v/v), about 0.1% to about 0.15% (v/v), about 0.1% to about0.2% (v/v), about 0.1% to about 0.25% (v/v), about 0.1% to about 0.3%(v/v), about 0.1% to about 0.4% (v/v), about 0.1% to about 0.5% (v/v),about 0.25% to about 0.5% (v/v), about 0.5% to about 1.5% (v/v), about0.75% to about 1.75% (v/v), about 1% to about 2% (v/v), about 1.25% toabout 2.25% (v/v), about 1.5% to about 2.5% (v/v), about 1.75% to about2.75% (v/v), about 2% to about 3% (v/v), about 2.25% to about 3.25%(v/v), about 2.5% to about 3.5% (v/v), about 2.75% to about 3.75% (v/v),about 3% to about 4% (v/v).

In other embodiments, the amount of ethanol present in the medium (forexample, a basal medium or a feed medium) can be any of about 0.001%(v/v), 0.005% (v/v), 0.01% (v/v), 0.011% (v/v), 0.012% (v/v), 0.013%(v/v), 0.014% (v/v), 0.015% (v/v), 0.016% (v/v), 0.017% (v/v), 0.018%(v/v), 0.019% (v/v), 0.02% (v/v), 0.021% (v/v), 0.022% (v/v), 0.023%(v/v), 0.024% (v/v), 0.025% (v/v), 0.026% (v/v), 0.027% (v/v), 0.028%(v/v), 0.029% (v/v), 0.03% (v/v), 0.031% (v/v), 0.032% (v/v), 0.033%(v/v), 0.034% (v/v), 0.035% (v/v), 0.036% (v/v), 0.037% (v/v), 0.038%(v/v), 0.039% (v/v), 0.04% (v/v), 0.041% (v/v), 0.042% (v/v), 0.043%(v/v), 0.044% (v/v), 0.045% (v/v), 0.046% (v/v), 0.047% (v/v), 0.048%(v/v), 0.049% (v/v), 0.05% (v/v), 0.051% (v/v), 0.052% (v/v), 0.053%(v/v), 0.054% (v/v), 0.055% (v/v), 0.056% (v/v), 0.057% (v/v), 0.058%(v/v), 0.059% (v/v), 0.06% (v/v), 0.061% (v/v), 0.062% (v/v), 0.063%(v/v), 0.064% (v/v), 0.065% (v/v), 0.066% (v/v), 0.067% (v/v), 0.068%(v/v), 0.069% (v/v), 0.07, % (v/v) 0.071, % (v/v) 0.072, % (v/v) 0.073%(v/v), 0.074% (v/v), 0.075% (v/v), 0.076% (v/v), 0.077% (v/v), 0.078%(v/v), 0.079% (v/v), 0.08% (v/v), 0.081% (v/v), 0.082% (v/v), 0.083%(v/v), 0.084% (v/v), 0.085% (v/v), 0.086% (v/v), 0.087% (v/v), 0.088%(v/v), 0.089% (v/v), 0.09% (v/v), 0.091% (v/v), 0.092% (v/v), 0.093%(v/v), 0.094% (v/v), 0.095% (v/v), 0.096% (v/v), 0.097% (v/v), 0.098%(v/v), 0.099% (v/v), 0.1% (v/v), 0.11% (v/v), 0.12% (v/v), 0.13% (v/v),0.14% (v/v), 0.15% (v/v), 0.16% (v/v), 0.17% (v/v), 0.18% (v/v), 0.19%(v/v), 0.2% (v/v), 0.21% (v/v), 0.22% (v/v), 0.23% (v/v), 0.24% (v/v),0.25% (v/v), 0.26% (v/v), 0.27% (v/v), 0.28% (v/v), 0.29% (v/v), 0.3%(v/v), 0.31% (v/v), 0.32% (v/v), 0.33% (v/v), 0.34% (v/v), 0.35% (v/v),0.36% (v/v), 0.37% (v/v), 0.38% (v/v), 0.39% (v/v), 0.4% (v/v), 0.41%(v/v), 0.42% (v/v), 0.43% (v/v), 0.44% (v/v), 0.45% (v/v), 0.46% (v/v),0.47% (v/v), 0.48% (v/v), 0.49% (v/v), 0.5% (v/v), 0.6% (v/v), 0.7%(v/v), 0.8% (v/v), 0.9% (v/v), 1% (v/v), 1.1% (v/v), 1.2% (v/v), 1.3%(v/v), 1.4% (v/v), 1.5% (v/v), 1.6% (v/v), 1.7% (v/v), 1.8% (v/v), 1.9%(v/v), 2% (v/v), 2.1% (v/v), 2.2% (v/v), 2.3% (v/v), 2.4% (v/v), 2.5%(v/v), 2.6% (v/v), 2.7% (v/v), 2.8% (v/v), 2.9% (v/v), 3% (v/v), 3.1%(v/v), 3.2% (v/v), 3.3% (v/v), 3.4% (v/v), 3.5% (v/v), 3.6% (v/v), 3.7%(v/v), 3.8% (v/v), 3.9% (v/v), or 4% (v/v) or more, inclusive of allvalues falling in between these percentages.

IV. METHODS OF THE INVENTION

A. Methods for Producing Recombinant Polypeptides

In some aspects, provided herein are methods for producing one or morerecombinant polypeptides from an engineered mammalian cell. The methodentails culturing the engineered mammalian cell in any of the mammaliancell culture medium compositions described herein under suitableconditions for the production of one or more recombinant polypeptides;and producing one or more recombinant polypeptides.

Culturing of recombinant mammalian cells for the production ofrecombinant polypeptides is well known in the art. The polypeptides thatcan be produced from the cell culture media according to the presentinvention are not limited. The term “polypeptide” as used hereinencompasses molecules composed of a chain of more than two amino acidsjoined by peptide bonds; molecules containing two or more such chains;molecules comprising one or more such chains being additionallymodified, e.g., by glycosylation. The term polypeptide is intended toencompass proteins.

Any polypeptide that can be expressed in a mammalian host cell may beproduced according to the present invention. After the polypeptide(s)has/have been produced in the media of the present invention it iseither extracellularly secreted, bound to the cells or remains in thecells, depending on the specific product and cell line used. Thepolypeptide product can be recovered from culture supernatant directlyor after lysis of the cells by standard procedures. In furtherembodiments, isolation and purification of recombinant polypeptides isperformed using standard techniques known in the art. These can include,without limitation, size exclusion chromatography, protein A affinitychromatography, anion exchange chromatography, cation exchangechromatography, mixed mode chromatography, or hydrophobic interactionchromatography.

One non-limiting class of polypeptides produced by the cell culturemedia according to the present invention is recombinant antibodies. Theterm “antibody” is used in the broadest sense and specifically coversmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), nanobodies modified antibodies, subunits of antibodies,antibody derivatives, artificial antibodies, combinations of antibodieswith proteins and antibody fragments sufficiently long to display thedesired biological activity. The monoclonal antibodies as used hereinmay be human antibodies or humanized antibodies. In one embodiment, therecombinant polypeptide is a monoclonal antibody selected from the groupconsisting of trastuzumab, pertuzumab, infliximab, adalimumab,bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab,daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.

However, polypeptides other than antibodies can also be produced usingcell cultures and the cell culture media according to the presentinvention, e.g. therapeutic polypeptides such as, without limitation,transmembrane proteins, receptors, hormones, growth factors, proteases,clotting and anti-clotting proteins, inhibitor proteins, interleukins,transport factors, fusion proteins and the like. As such, anothernon-limiting class of polypeptides produced in the cell culture mediaaccording to the present invention is therapeutic polypeptides. In oneembodiment, the recombinant polypeptide is a therapeutic polypeptideselected from the group consisting of abatacept, abobotulinumtoxinA,aflibercept, agalsidase beta, albiglutide, aldesleukin, alglucosidasealfa, alteplase, cathflo activase, anakinra, asfotase alfa,asparaginase, becaplermin, belatacept, collagenase, collagenaseClostridium histolyticum, darbepoetin alfa, denileukin, diftitox,dornase alfa, dulaglutide, ecallantide, elosulfase alfa, epoetin alfa,etanercept, filgrastim, galsulfase, glucarpidase, idursulfase,incobotulinumtoxinA, interferon alfa-2b, interferon alfa-n3, interferonbeta-1a, interferon beta-1a, interferon beta-1b, interferon beta-1b,interferon gamma-1b, laronidase, methoxy polyethylene glycol-epoetinbeta, metreleptin, ocriplasmin, onabotulinumtoxinA, oprelvekin,palifermin, parathyroid hormone, pegaspargase, pegfilgrastim,peginterferon alfa-2a, peginterferon alfa-2a, ribavirin, peginterferonalfa-2b, peginterferon beta-1a, pegloticase, rasburicase, reteplase,rilonacept, rimabotulinumtoxinB, romiplostim, sargramostim, sebelipase,tbo-filgrastim, tenecteplase, and ziv-aflibercept.

In some embodiments, it may be advantageous to change the temperatureduring the course of culturing the mammalian cells and include one ormore temperature shifts that are initiated at certain time points. Achange/shift in the temperature does not refer to spontaneousfluctuations in the temperature, but to changes in temperature of atleast 1° C. or alternatively at least 2° C., 3° C., 4° C., 5° C., 6° C.,7° C., 8° C., 9° C., or 10° C. that are intended, and where the secondtemperature is being maintained for at least one day. A change/shift canbe implemented by altering the temperature set point of the culture. Thetiming is dependent on either the growth state of the culture, apredetermined number of days after the start of the culture or themetabolic needs of the cells. Thus, the temperature may be shifted in aperiod of about 1 to 10 days after starting the culture. In someembodiments, a temperature shift is done during the growth phase of thecells or towards the end of this phase and prior to the beginning of theproduction phase of the recombinant protein. Depending on the culturevessel volume, the change may occur rapidly or more slowly and lastsseveral hours, in one example such a shift in temperature is implementedduring the growth phase of the culture when the density is between about40 and about 90% of the maximal density. In one example the firsttemperature is between about 33 and about 38° C., while in otherexamples the first temperature is between about 35 and about 37° C. Thesecond temperature is between about 28 and about 36° C., oralternatively between about 29 and about 35° C.

In another embodiment of the present invention, it may be advantageousto change the pH during the course of the culturing of the mammaliancells by including one or more pH shifts. In further aspects of theinvention shifts in temperature may also be combined with one or moreshifts in the pH. In other embodiments, the pH of the culture medium(such as any mammalian cell culture media described herein) can varyduring the course of fermentation, such as from between about pH 6.7 toabout pH 7.3, such as any pH of about 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or7.3.

The cell culture medium according to the present invention can be usedin various mammalian cell culture processes. Cultivation of cells can becarried out in adherent culture, for instance in monolayer culture or insuspension culture. Large scale cultivation of cells can be used forinstance by the various fermentation processes established in industrialbiotechnology using, for example, small or large scale bioreactors.Continuous and discontinuous cell culture processes can be utilizedusing the cell culture media according to the present invention. Otherknown reactor technologies, e.g. perfusion technologies or the like canbe also utilized.

Batch processes are also a possible embodiment. Batch cell cultureincludes fed-batch culture or simple batch culture. The term “fed-batchcell culture” refers to cell culture wherein mammalian cells and cellculture medium are supplied to the culturing vessel initially andadditional culture nutrients are fed continuously or in discreteincrements to the culture during the culturing process with or withoutperiodic cell and/or product harvest before termination of the culture.The term “simple batch culture” relates to a procedure in which allcomponents for cell culturing including the mammalian cells and the cellculture medium are supplied to the culturing vessel at the start of theculturing process.

According to one embodiment of the present invention feeding of thecultures is done in a fed-batch process. Such feeding is beneficial forthe cells to replace media components and nutrients that are depleted inthe media during the culture process. Typically feed solutions compriseamino acids, at least one carbohydrate as an energy source, traceelements, vitamins or specific ions. The feed solutions are addeddepending on the needs of the cells, which are either based on apredetermined schedule that has been determined for the particular cellline or cell clone and product or measured during the culture process.It is particularly advantageous to use concentrated feed solutions inorder to avoid large volume increase and dilution of the media. In someembodiments it may also be useful to have at least two different feedsolutions. This allows independent dosing of two or more differentgroups of nutrients and components to the cells and thus a betteradjustment of the feeding conditions concerning optimal supply ofcertain nutrients.

The products obtained from such cell culture processes can be used forthe preparation of pharmaceutical compositions. The term “pharmaceuticalcomposition” indicates a composition suitable or adapted foradministration to a mammal, for example, a human. In addition, theprotein(s) according to the invention can be administered together withother components of biologically active agents such as pharmaceuticallyacceptable surfactants, recipients, carriers, diluents and vehicles. Infurther embodiments, recombinant polypeptides produced in accordancewith any of the methods of the present invention can be lyophilized andformulated for intravenous, parenteral, or subcutaneous administration.

In some embodiments, culturing of mammalian cells in any of the mediadisclosed herein in accordance with any of the methods disclosed hereinresults in an increased recombinant protein titer in comparison toproduction by mammalian cells that are not cultured in any of the mediadisclosed herein in accordance with any of the methods disclosed herein.In some embodiments, the method increases the titer of the recombinantpolypeptide by any of about 1% to about 2%, about 1% to about 3%, about1% to about 4%, about 1% to about 5%, about 2.5% to about 5%, about 2.5%to about 7.5%, about 5% to about 7.5%, about 5% to about 10%, about 1 toabout 15%, about 7.5% to about 12.5%, about 7.5% to about 15%, about7.5% to about 17.5%, about 10% to about 12.5%, about 10% to about 15%,about 10% to about 17.5%, about 10% to about 20%, about 12.5% to about15%, about 12.5% to about 17.5%, about 12.5% to about 20%, about 12.5%to about 22.5%, about 15% to about 17.5%, about 15% to about 20%, about15% to about 22.5%, about 15% to about 25%, about 17.5% to about 20%,about 17.5% to about 22.5%, about 17.5% to about 25%, about 17.5% toabout 27.5%, about 20% to about 22.5%, about 20% to about 25%, about 20%to about 27.5%, about 20% to about 30%, about 22.5% to about 25%, about22.5% to about 27.5%, about 22.5% to about 30%, about 22.5% to about32.5%, about 25% to about 27.5%, about 25% to about 30%, about 25% toabout 32.5%, or about 25% to about 35%, or at least any of 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, inclusive of values fallingin between these percentages, compared to the titer of recombinantpolypeptides produced by mammalian cells that are not cultured in themedia disclosed herein and in accordance with the methods describedherein.

B. Methods for Modulating the Glycosylation Profile of RecombinantPolypeptides

Glycan species have been shown to significantly influencepharmacokinetics (PK) and pharmacodynamics (PD) of therapeutic proteinssuch as mAbs. Accordingly, in other aspects of the present invention,provided herein are methods for modulating the glycosylation profile ofone or more recombinant polypeptides produced by a geneticallyengineered mammalian cell. In some embodiments, the method involvesculturing a mammalian cell in any of the cell culture media describedherein (such as a lithium-containing, an ethanol-containing, and/or afatty acid-containing cell culture media) under suitable conditions forthe production of said one or more recombinant polypeptides; andproducing the one or more recombinant polypeptides, wherein the one ormore recombinant polypeptides has a modulated glycosylation profilecompared to recombinant polypeptides produced by mammalian cells thatare not cultured in any of the cell culture media described herein. Insome embodiments, “modulating the glycosylation profile” refers toincreasing the relative percentage of a particular glycan species on therecombinant polypeptide. However, in other embodiments, the phrase“modulating the glycosylation profile” refers to decreasing the relativepercentage of a particular glycan species on the recombinantpolypeptide. Any relative percentage of any glycan species capable ofaddition to a recombinant polypeptide can be altered when produced inaccordance with the methods disclosed herein.

In some embodiments, culturing mammalian cells that have beengenetically engineered to produce recombinant polypeptides in any mediumdescribed herein (such as a lithium-containing, an ethanol-containing,and/or a fatty acid-containing cell culture media) modulated the amountof one or more of glycan species. Examples of glycan species include,without limitation, mannose-9-N-acetylglycosamine-2 (Man9),mannose-8-N-acetylglycosamine-2 (Man8), mannose-7-N-acetylglycosamine-2(Man7), mannose-6-N-acetylglycosamine-2 (Man6),mannose-5-N-acetylglycosamine-2 (Man5), mannose-3-N-acetylglucosamine-2(Man3), mannose-3-N-acetylglucosamine-3, mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-3-fucose,mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1 (G1),mannose-3-N-acetylglucosamine-4-galactose-2 (G2),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F),mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose-1-N-acetylneuraminic-1(G1F-NANA),mannose-3-N-acetylglucosamine-4-galactose-2-fucose-1-N-acetylneuraminic-1(G2F-NANA), and/ormannose-3-N-acetylglucosamine-4-galactose-2-fucose-1-N-acetylneuraminic-2(G2F-2NANA) bound to the recombinant polypeptide. FIG. 1 depicts aschematic representation of major N-glycan linked species.

Alteration of glycan species on recombinant polypeptides can beseparated and measured in accordance with any means known in the artsuch as, without limitation, high performance liquid chromatography(HPLC), normal-phase HPLC (NP), hydrophilic interaction liquidchromatography (HILIC), ion-exchange HPLC (IEX), weak anion exchangeHPLC (WAX-HPLC), zwitterionic sulfobetaine (ZIC-HILIC), porous graphitecarbon HPLC (PGC), capillary electrophoresis laser induced fluorescent(CE-LIF), matrix-assisted laser desorption/ionization time of flight(MALDI-TOF), electrospray ionization mass spectrometry (ESI-MS), liquidchromatography mass spectrometry (LC-MS) and tandem mass spectrometry(MS/MS).

In some embodiments, the culturing methods provided herein reduce aspecific glycan species normalized or relative to the total glycan poolon a recombinant polypeptide. For example, in some embodiments, themethod modulates (e.g. decreases) one or more specific glycan species onthe recombinant polypeptides by any of about 1% to about 2%, about 1% toabout 3%, about 1% to about 4%, about 1% to about 5%, about 2.5% toabout 5%, about 2.5% to about 7.5%, about 5% to about 7.5%, about 5% toabout 10%, about 1 to about 15%, about 7.5% to about 12.5%, about 7.5%to about 15%, about 7.5% to about 17.5%, about 10% to about 12.5%, about10% to about 15%, about 10% to about 17.5%, about 10% to about 20%,about 12.5% to about 15%, about 12.5% to about 17.5%, about 12.5% toabout 20%, about 12.5% to about 22.5%, about 15% to about 17.5%, about15% to about 20%, about 15% to about 22.5%, about 15% to about 25%,about 17.5% to about 20%, about 17.5% to about 22.5%, about 17.5% toabout 25%, about 17.5% to about 27.5%, about 20% to about 22.5%, about20% to about 25%, about 20% to about 27.5%, about 20% to about 30%,about 22.5% to about 25%, about 22.5% to about 27.5%, about 22.5% toabout 30%, about 22.5% to about 32.5%, about 25% to about 27.5%, about25% to about 30%, about 25% to about 32.5%, or about 25% to about 35%,or at least any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%,inclusive of values falling in between these percentages, compared tothe amount of the glycan species of recombinant polypeptides produced bymammalian cells that are not cultured in the media disclosed herein(such as a lithium-containing, an ethanol-containing, and/or a fattyacid-containing cell culture media) and in accordance with the methodsdescribed herein. In some embodiments, the glycan species is Man5, G0,or G0F.

In some embodiments, the culturing methods provided herein increase aspecific glycan species normalized or relative to the total glycan poolon a recombinant polypeptide. For example, in yet other embodiments, themethod increases one or more specific glycan species by any of about 1%to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% toabout 5%, about 2.5% to about 5%, about 2.5% to about 7.5%, about 5% toabout 7.5%, about 5% to about 10%, about 1 to about 15%, about 7.5% toabout 12.5%, about 7.5% to about 15%, about 7.5% to about 17.5%, about10% to about 12.5%, about 10% to about 15%, about 10% to about 17.5%,about 10% to about 20%, about 12.5% to about 15%, about 12.5% to about17.5%, about 12.5% to about 20%, about 12.5% to about 22.5%, about 15%to about 17.5%, about 15% to about 20%, about 15% to about 22.5%, about15% to about 25%, about 17.5% to about 20%, about 17.5% to about 22.5%,about 17.5% to about 25%, about 17.5% to about 25%.7, about 20% to about22.5%, about 20% to about 25%, about 20% to about 27.5%, about 20% toabout 30%, about 22.5% to about 25%, about 22.5% to about 27.5%, about22.5% to about 30%, about 22.5% to about 32.5%, about 25% to about27.5%, about 25% to about 30%, about 25% to about 32.5%, or about 25% toabout 35%, or at least any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, or 40%, inclusive of values falling in between these percentages,compared to the amount of the glycan species of recombinant polypeptidesproduced by mammalian cells that are not cultured in the media disclosedherein (such as a lithium-containing, an ethanol-containing, and/or afatty acid-containing cell culture media) and in accordance with themethods described herein.

In some embodiments, culturing mammalian cells genetically engineered toproduce one or more recombinant polypeptides in any of the mediadisclosed herein according the methods described herein modulates (e.g.reduces) the amount of terminal mannose glycan species in comparison tothe total sum of all glycan species on the polypeptide. As used herein,the phrase “terminal mannose glycan species” refers to one or more ofmannose-5-N-acetylglycosamine-2 (Man5), mannose-6-N-acetylglycosamine-2(Man6), mannose-7-N-acetylglycosamine-2 (Man7),mannose-8-N-acetylglycosamine-2 (Man8) and/ormannose-9-N-acetylglycosamine-2 (Man9) moieties.

As such, for recombinant polypeptides produced in accordance with any ofthe methods described herein using any of the cell culture mediadisclosed herein (such as a lithium-containing, an ethanol-containing,and/or a fatty acid-containing cell culture media), the ratio of theterminal mannose glycan species to the total sum of glycan species onthe polypeptide can be modulated (e.g., decreased) by about 30% to about70%, about 35% to about 65%, about 40% to about 60%, about 45% to about55%, or about 47.5% to about 52.5%, such as any of about 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% or more.

In some embodiments, culturing mammalian cells genetically engineered toproduce one or more recombinant polypeptides in any of theethanol-containing media disclosed herein according the methodsdescribed herein modulates the amount of any one of G1F, G2F, and/or G0Fglycan species in comparison to the total sum of all glycan species onthe polypeptide. As such, for recombinant polypeptides produced inaccordance with any of the methods described herein using any of thecell culture media disclosed herein (such as a lithium-containing, anethanol-containing, and/or a fatty acid-containing cell culture media),the ratio any one of G1F, G2F, and/or G0F glycan species to the totalsum of glycan species on the polypeptide can be modulated by about 30%to about 70%, about 35% to about 65%, about 40% to about 60%, about 45%to about 55%, or about 47.5% to about 52.5%, such as any of about 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% or more.

C. Methods for Modulating High or Low Molecular Weight Species

In further aspects, provided herein are methods for modulating (e.g.reducing) the amount of high or low molecular weight species of one ormore recombinant polypeptides produced by an engineered mammalian cell.The method entails culturing a mammalian cell in any of the cell culturemedia described herein (such as a lithium-containing, anethanol-containing, and/or a fatty acid-containing cell culture media)under suitable conditions for the production of said one or morerecombinant polypeptides; and producing the one or more recombinantpolypeptides, wherein the recombinant polypeptides have reduced amountsof high or low molecular weight species compared to recombinantpolypeptides produced by recombinant mammalian cells that are notcultured in any of the cell culture media of the present invention.

“High molecular weight species” (HMWS) as used in the context of thepresent invention means the development of any particle which consistsof more than one subunit of a recombinant polypeptide, also includingoligomers, such as e.g. dimers, trimers, tetramers, pentamers and thelike. HMWS can also consist of more than 2 subunits, such as any of 3,4, 5, 6, 7, 8, or more. HMWS can be of different sizes, which have amass greater than that of the fully assembled recombinant polypeptide.

“Low molecular weight species” (LMWS) as used in the context of thepresent invention means the development of any particle which consistsof one or more subunit of a recombinant polypeptide, also includingoligomers, such as e.g. dimers, trimers, tetramers, pentamers and thelike. LMWS can comprise of polypeptide fragments that are not completelyassembled and/or folded. LMWS can be of different sizes, which have amass less than that of the fully assembled recombinant polypeptide.

The amount of high or low molecular weight species of the recombinantpolypeptides can be measured in accordance with any means known in theart such as, without limitation, size exclusion chromatography (SEC),analytical ultra-centrifugation (AUC), dynamic or staticlight-scattering spectroscopy (DLS), differential scanning calorimetry(DSC) or asymmetric flow field flow fractionation (AF4).

In some embodiments, the cell culture methods disclosed herein modulate(e.g. decrease) the amount of HMW species compared to total detectedpolypeptide molecular weight variants by any of about 1% to about 2%,about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about2.5% to about 5%, about 2.5% to about 7.5%, about 5% to about 7.5%,about 5% to about 10%, about 1 to about 15%, about 7.5% to about 12.5%,about 7.5% to about 15%, about 7.5% to about 17.5%, about 10% to about12.5%, about 10% to about 15%, about 10% to about 17.5%, about 10% toabout 20%, about 12.5% to about 15%, about 12.5% to about 17.5%, about12.5% to about 20%, about 15% to about 17.5%, about 15% to about 20%,about 17.5% to about 20%, about 17.5% to about 22.5%, about 17.5% toabout 25%, about 20% to about 22.5%, about 20% to about 25%, about 22.5%to about 25%, or at least any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, or 25% or more, inclusive of values falling in between thesepercentages, compared to the amount of HMW species of recombinantpolypeptides produced by mammalian cells that are not cultured in amedia disclosed herein (such as a lithium-containing, anethanol-containing, and/or a fatty acid-containing cell culture media)and in accordance with the methods described herein.

In other embodiments, the cell culture methods disclosed herein modulate(e.g. decrease) the amount of LMW species compared to total detectedpolypeptide molecular weight variants by any of about 1% to about 5%,about 2.5% to about 7.5%, about 5% to about 7.5%, about 5% to about 10%,about 7.5% to about 12.5%, about 7.5% to about 15%, about 7.5% to about17.5%, about 10% to about 15%, about 10% to about 17.5%, about 10% toabout 20%, about 12.5% to about 17.5%, about 12.5% to about 20%, about12.5% to about 22.5%, about 15% to about 20%, about 15% to about 22.5%,about 15% to about 25%, about 17.5% to about 22.5%, about 17.5% to about25%, about 17.5% to about 27.5%, about 20% to about 25%, about 20% toabout 27.5%, about 20% to about 30%, about 22.5% to about 27.5%, about22.5% to about 30%, about 22.5% to about 32.5%, about 25% to about 30%,about 25% to about 32.5%, about 25% to about 35%, about 27.5% to about37.5%, about 30% to about 40%, about 32.5% to about 44.5%, about 35% toabout 45%, about 37.5% to about 47.5%, about 40% to about 50%, about42.5% to about 52.5%, about 45% to about 55%, about 47.5% to about57.5%, about 50% to about 60%, about 52.5% to about 62.5%, about 55% toabout 65%, about 57.5% to about 67.5%, about 60% to about 70%, about62.5% to about 72.5%, or about 65% to about 75% or at least any of 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75% or more, inclusive of values falling in between thesepercentages, compared to the amount of LMW species of recombinantpolypeptides produced by mammalian cells that are not cultured in amedia disclosed herein (such as a lithium-containing, anethanol-containing, and/or a fatty acid-containing cell culture media)and in accordance with the methods described herein.

D. Methods for Modulating the Amount of Acidic or Basic Charge Species

In further aspects, provided herein are methods for modulating (e.g.reducing) the amount of acidic or basic charge species of one or morerecombinant polypeptides produced by an engineered mammalian cell. Themethod entails culturing a mammalian cell in any of the cell culturemedia described herein under suitable conditions for the production ofsaid one or more recombinant polypeptides; and producing the one or morerecombinant polypeptides, wherein the recombinant polypeptides havereduced amounts of acidic or basic charge species compared torecombinant polypeptides produced by mammalian cells that are notcultured in any of the cell culture media described herein (such as alithium-containing, an ethanol-containing, and/or a fattyacid-containing cell culture media).

As used herein, or “acidic charge species” or “acidic charge variants”refer to the percentage of recombinantly produced proteins in amammalian cell culture that bear acidic charges compared to the totalpolypeptide charge variants of the total population of recombinantlyproduced proteins. Similarly, “basic charge species” or “basic chargevariants,” as used herein, refer to the percentage of recombinantlyproduced proteins in a mammalian cell culture that bear basic chargescompared to the total polypeptide charge variants of the totalpopulation of recombinantly produced proteins.

In some embodiments, the cell culture methods disclosed herein modulate(e.g. decrease) the amount of acidic charge species compared to totaldetected polypeptide charge variants by any of about 1% to about 2%,about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about2.5% to about 5%, about 2.5% to about 7.5%, about 5% to about 7.5%,about 5% to about 10%, about 1 to about 15%, about 7.5% to about 12.5%,about 7.5% to about 15%, about 10% to about 12.5%, about 10% to about15%, or about 12.5% to about 15%, or at least any of 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or more, inclusive ofvalues falling in between these percentages, compared to the amount ofacidic charge species of recombinant polypeptides produced by mammaliancells that are not cultured in a media disclosed herein (such as alithium-containing, an ethanol-containing, and/or a fattyacid-containing cell culture media) and in accordance with the methodsdescribed herein.

In other embodiments, the cell culture methods disclosed herein modulate(e.g. decrease) the amount of basic charge species compared to totaldetected polypeptide charge variants by any of about 1% to about 2%,about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about2.5% to about 5%, about 2.5% to about 7.5%, about 5% to about 7.5%,about 5% to about 10%, about 1 to about 15%, about 7.5% to about 12.5%,about 7.5% to about 15%, about 10% to about 12.5%, about 10% to about15%, or about 12.5% to about 15%, or at least any of 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or more, inclusive ofvalues falling in between these percentages, compared to the amount ofbasic charge species of recombinant polypeptides produced by mammaliancells that are not cultured in a media disclosed herein (such as alithium-containing, an ethanol-containing, and/or a fattyacid-containing cell culture media) and in accordance with the methodsdescribed herein.

V. KITS

In addition, the present invention includes a kit for culturingmammalian cells in accordance with any of the methods disclosed herein.The kits can contain one or more of a mammalian cell culture basalmedium and/or a mammalian cell culture feed medium. Further, the kitscan additionally contain one or more of a source of lithium ions,ethanol, and one or more fatty acids. The kit can also include writteninstructions for using the kit, such as instructions for making any ofthe mammalian cell culture media disclosed herein as well asinstructions for using a medium to culture mammalian cells (such as,culturing mammalian cells genetically engineered for the production ofone or more recombinant polypeptides).

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example 1: Addition of Lithium to Cell Culture Medium ImprovesProduction and Quality of Recombinant Polypeptides

This Example shows the effect of lithium addition to culture media withrespect to production of a recombinant antibody.

I. Determination of Cytotoxicity

Lithium chloride cytotoxicity to CHO.DXB11 cells was determined by asingle dose bolus spike. 4 mL of CHO cells at 6 e6/mL cellular densitywere seeded into 6 well deep plates, mixed at 150 RPM, and spiked withlithium chloride to a working concentration range of 5 mM to 250 mM.Cell growth rate and cell viability were negatively affected when singledose spiking of lithium chloride exceeded 10 mM.

II. Addition of Lithium Using Microscale Bioreactors for MonoclonalAntibody Production

To determine if LiCl altered mAb properties it was incorporated intofully defined and synthetic feed media and fed daily in a fed-batchprocess using micro- (15 mL working volume) bioreactors.

Materials and Methods

Monoclonal antibody 1 (mAb 1) is a monoclonal antibody produced usingrecombinant DNA technology. The expression vector is fully syntheticwith both heavy- and light-chain gene sequences regulated by strongconstitutive promoters. Gene sequences were confirmed by DNA sequencing.The expression vector was linearized by restriction enzymes and stablytransfected into CHO.DXB11 cells by electroporation.

Single cell cloning was performed after gene amplification. Selectsingle cell clones were expanded and cryopreserved.

Titer was measured using a Thermo Ultimate 3000 Series HPLC with thedetection wavelength set at 280 nm. Mobile phase A contained sodiumphosphate and sodium chloride with mobile phase B containing acetic acidand magnesium chloride. An Applied Biosystems POROS A20 column was usedand analysis was performed using the Thermo Chromeleon software.Purified antibody was used as the standard for quantitation.

Glycosylation species were measured with the Thermo Ultimate 3000 SeriesHPLC and the Thermo Q Exactive Mass Spectrometer. Mobile phase Aconsists of formic acid and trifluoroacetic acid in water with mobilephase B consisting of formic acid and trifluoroacetic acid inacetonitrile. An Agilent PLRP-S 1000A 5 μM column was used and analysiswas performed using the Thermo Excalibur software in conjunction withthe Thermo Protein Deconvolution software.

LiCl was fed daily to increase working bioreactor concentrations between0.11 mM and 1.11 mM. Calculated cumulative cell culture concentration(concentration in vessel at time of cell harvest) of lithium rangedbetween 1 mM 10 mM. Bioreactor physical conditions were maintained at apH of 7.0±0.2, dissolved oxygen at 30% air saturation and temperaturesof 37° C. or 35° C. for logarithmic cellular growth phase and shifted to31° C. during cellular production phase (temperature was shifted one daybefore max viable cell density was reached). Basal and feed media arefully synthetic and chemically defined containing no animal derivedcomponents.

Results

Lithium was shown to increase antibody titer at the high end of theconcentrations used (FIG. 2). Higher doses of lithium increased titerover 21% compared to baseline. Furthermore, higher concentrations oflithium reduced Man5 glycan species by over 13% (FIG. 2).

III. Addition of Lithium Using Microscale Bioreactors for MonoclonalAntibody Production in a Design of Experiment (DOE) Study

Lithium was used in design of experiments (DOE) created with custom DOEdesign using JMP statistical software (World WideWeb.jmp.com/en_us/home.html). Lithium was fed daily to increasebioreactor working concentrations by 0.5 mM or 1 mM in a 12 dayfed-batch process. A response model was created using standard leastsquares to determine the significance of response from lithiumsupplementation. Potential interactions with other supplementedcomponents were also considered.

FIG. 3 depicts the results of the DOE analysis. Titer, Man5%, G0%, HMW%, LMW %, Acidic % and Basic % species were assessed. The predictionprofile shows how the model changes as individual factors are varied inorder to gauge the model's sensitivity to changes in those factors. Asshown below in Table 1, addition of 1 mM lithium increased titer by over20%, decreased Man5 glycan species by over 6%, decreased high and lowmolecular weight species by over 9% and 46%, respectively, and alsodecreased acidic and basic charge variant species by over 9% and 3%,respectively.

TABLE 1 Change in response values extracted from the prediction profileof Titer, Man5%, G0%, HMW %, LMW %, Acidic % and Basic % species. TiterMan5% G0% HMW % LMW % Acidic % Basic % 0.5 mM +10.7% −3.1% +5.2% −4.5%−23.0% −4.5% −1.5% Lithium 1.0 mM +21.3% −6.3% +10.4% −9.1% −46.0% −9.1%−3.0% Lithium

IV. Addition of Lithium Using Microscale Bioreactors for MonoclonalAntibody Production in a Design of Experiment (DOE) Study UsingAlternate Variables

Lithium was used in design of experiment, as described above, usingalternative process conditions. Physical conditions were altered bychanging temperature and pH set points. Feed formulations were modifiedto include additional amino acids and trace elements. Lithium was feddaily to increase bioreactor working concentrations by 0.5 mM or 1 mM ina 13 day fed-batch process. A response model was created using standardleast squares in order to determine the significance of response fromlithium supplementation. Potential interactions with other supplementedcomponents were also considered.

Results are shown in FIG. 4. Titer, Man5%, G0%, G0F %, G1F %, G2F %, HMW%, LMW %, Acidic % and Basic % species were assessed. The change inresponse values extracted from the prediction profile is shown in Table2 below. At 1 mM concentration, lithium increased titer by almost 17%,decreased Man5 glycan variants by more the 26%, decreased high and lowmolecular weight species by more than 12% and more than 61%,respectively, and decreased acidic and basic charge variants by morethan 4% compared to baseline.

TABLE 2 Change in response values extracted from the prediction profileof Titer, Man5%, G0%, G0F %, G1F %, G2F %, HMW %, LMW %, Acidic % andBasic % species. Titer Man5% G0% G0F % G1F % G2F % HMW % LMW % Acidic %Basic % 0.5 mM +8.5% −13.2% −2.0% −0.5% +0.8% −0.3% −6.1% −30.9% −2.4%−2.3% Lithium 1.0 mM +16.9% −26.4% −4.0% −0.9% +1.6% −0.6% −12.2% −61.8%−4.8% −4.5% Lithium

DOE experimental results from the two analyses described above werecombined into one prediction model using a total of 48 independentbioreactor conditions. By combining data sets, confidence intervalstighten and p-values decrease leading to a higher significance ofresponse. The prediction response profile for this combined data isshown in FIG. 5 and the change in response values extracted from theprediction profile are shown below in Table 3. As shown, at 1 mMconcentration, lithium increased titer by almost 20%, decreased Man5glycan variants by more than 14%, decreased high and low molecularweight species by more than 10% and more than 52%, respectively, anddecreased acidic and basic charge variants by more than 6% and 3%,respectively.

TABLE 3 Change in response values extracted from the prediction profileof Titer, Man5%, G0%, G0F %, G1F %, G2F %, HMW %, LMW %, Acidic % andBasic % species. Titer Man5% G0% G0F % G1F % G2F % HMW % LMW % Acidic %Basic % 0.5 mM +9.7% −7.3% +1.6% +2.6% −2.6% −5.8% −5.2% −26.2% −3.4%−1.8% Lithium 1.0 mM +19.4% −14.6% +3.2% +5.3% −5.1% −11.5% −10.4%−52.4% −6.9% −3.6% Lithium

V. Use of Lithium-Supplemented Media for Production of a DifferentMonoclonal Antibody

Monoclonal antibody 2 (mAb 2) is a monoclonal antibody produced usingrecombinant DNA technology. The expression vector is fully syntheticwith both heavy- and light-chain gene sequences regulated by strongconstitutive promoters. Gene sequences were confirmed by DNA sequencing.The expression vector was linearized by restriction enzymes and stablytransfected into CHO.DG44 cells by electroporation. Two rounds of singlecell cloning were performed. Single cell clones were expanded andcryopreserved.

Lithium was tested using a different CHO host (CHO DG44) which producesmonoclonal antibody #2 (mAb2). Cell culture and monoclonal antibodyproduction was performed as described above. Cells were grown inmicroscale bioreactors and lithium was fed daily to increase bioreactorconcentrations by 0.11 mM (Low Li), 0.44 mM (Medium Li) and 1.11 mM(High Li) in a 13 day fed-batch process. Results indicated thatsupplementing lithium into the cell culture media reduced Man6% glycanlevels (FIG. 6) and Man5% glycan levels (FIG. 6) by 29% and 12%,respectively over baseline. Further, higher lithium concentrationsreduced high molecular weight species 24% over baseline (FIG. 6).

Example 2: Addition of Ethanol to Cell Culture Medium ImprovesProduction of Recombinant Polypeptides

Previously, an ethanol-based cholesterol supplement was developed inorder to cultivate cholesterol-dependent NSO cells in a linear,low-density polypropylene-based disposable bioreactor system. Thisethanol-based supplement contained cholesterol, oleic acid and linoleicacid (Tao et al., Biotechnol Lett., 2012; 34(8):1453-8). This Exampleinvestigated the use of ethanol as a solubilizing agent for variousfatty acids for the production of mAb products.

I. Determination of Cytotoxicity

Ethanol cytotoxicity to CHO.DXB11 cells was determined by daily feeddosing. 4 mL of CHO cells at 3 e6/mL cellular density were seeded into 6well deep plates, mixed at 150 RPM, and fed once daily with ethanolranging from 0.1% to 0.4% volume to volume (v/v). Cell growth rate andcell viability were negatively affected when daily ethanol feedingexceeded 0.3% v/v.

II. Addition of Ethanol Using Microscale Bioreactors for MonoclonalAntibody Production

To determine if ethanol altered mAb properties it was incorporated intofully defined and synthetic feed media and fed daily in a fed-batchprocess using micro- (15 mL working volume) bioreactors.

Materials and Methods:

Antibody production was performed as described above for mAb 1.

Cell line A is a CHO.DXB11 derived clone producing mAb 1. Cell line B isa different CHO.DXB11 derived clone producing mAb1. The expressioncassette is identical between the two cell lines.

Ethanol was fed daily 0.0% or 0.057% v/v using microscale bioreactorsand two different cellular clones producing mAb1. Calculated cumulativecell culture concentration (concentration in vessel at time of cellharvest) of ethanol ranged between 0.0%-0.51% v/v. Bioreactor physicalconditions were maintained at a pH of 7.0±0.2, dissolved oxygen at 30%air saturation and a temperature of 35° C. during logarithmic cellulargrowth phase and shifted to 31° C. during cellular production phase(temperature was shifted one day before max viable cell density wasreached). Basal and feed media were fully synthetic and chemicallydefined containing no animal derived components.

Results

Ethanol supplementation was observed to alter glycosylation profile,alter high molecular weight and low molecular weight species and alteracidic and basic charge species. As shown in FIG. 7 and FIG. 8, ethanoldecreased overall Man5% glycan species in both cell lines used, whereasthe effect on G0% and G0F % glycan species varied with respect to thecell line (FIG. 7 and FIG. 8). Further, ethanol increased overall G1F %and G2F % glycan species varied with respect to both cell lines used(FIG. 7 and FIG. 8). Moreover, ethanol decreased the relative amount ofacidic charge variants while slightly increasing the relative percentageof basic charge variants when compared to the total detected chargespecies variants for both cell lines tested (FIG. 7 and FIG. 8).

III. Addition of Ethanol Using Microscale Bioreactors for MonoclonalAntibody Production in a Design of Experiment (DOE) Study

Ethanol was used in design of experiments (DOE) created with custom DOEdesign using JMP statistical software (World WideWeb.jmp.com/en_us/home.html). A response model was created usingstandard least squares to determine the significance of response fromethanol supplementation. Potential interactions with other supplementedcomponents were also considered. Ethanol was fed daily between 0.0% and0.1% using microscale bioreactors. FIG. 9 is a prediction responseprofile for Titer, Man5%, G0%, Acidic % and Basic % species while thechange in response values for each of these is shown depicted in Table 4below.

TABLE 4 Change in response values extracted from the prediction profileof Titer, Man5%, G0%, Acidic % and Basic % species. Ethanol was feddaily to increase bioreactor working concentrations by 0.05% and 0.1%v/v in a 12 day fed-batch process. Titer Man5% G0% Acidic % Basic %0.05% v/v +2.0% −1.9% −0.7% −4.7% +1.0% Ethanol 0.1% v/v +3.9% −3.8%−1.3% −9.3% +1.9% Ethanol

IV. Addition of Ethanol Using Bench-Scale Bioreactors

Materials and Methods:

Antibody production was performed as described above for mAb 1.

Ethanol was fed daily 0.072% (Low Ethanol) or 0.144% (High Ethanol) v/v.Calculated cumulative cell culture concentration (concentration invessel at time of cell harvest) of ethanol ranged between 0.79%-1.58%v/v. Bioreactor physical conditions were maintained at a pH of 7.0±0.2,dissolved oxygen maintained at 30% of air saturation, temperature of 35°C. during logarithmic cellular growth phase and shifted to 31° C. duringcellular production phase (temperature was shifted one day before maxviable cell density was reached). Basal and feed media were fullysynthetic and chemically defined containing no animal derivedcomponents.

Results

As shown in FIG. 10, high ethanol concentration was associated with lessMan5 and G0 glycan species, greater G1F and G2F glycan species, and lesshigh and low molecular weight species.

V. Addition of Ethanol Using Bench-Scale Bioreactors and Two DifferentFeed Formulations

Materials and Methods:

Antibody production was performed as described above for mAb 1.

Ethanol was fed daily between 0.0% and 0.018% v/v. Calculated cumulativecell culture concentration (concentration in vessel at time of cellharvest) of ethanol ranged between 0.0%-0.2% v/v. Bioreactor physicalconditions were maintained at a pH of 7.0±0.2 shifted to 6.8±0.1,dissolved oxygen maintained at 30% air saturation and a constanttemperature of 35° C. Basal and feed media were fully synthetic andchemically defined containing no animal derived components.

The same basal media was used in formulation #1 and #2. Feed formulation#1 and #2 were similar in composition containing the same type andamount of amino acids and vitamins. Salt and metal ion type andconcentration were slightly varied. Both feeds were fully synthetic andchemically defined containing no animal derived components.

Results

As shown in FIG. 11, ethanol supplementation into Formulation 1 resultedin increased Man5, G1F, and G2F glycan species as well as basic chargevariants. In contrast, this combination resulted in less G0 and G0Fglycan species as well as less acidic charge variants. Ethanolsupplementation of Formulation 2 resulted in decreased Man5, G0, and G1Fglycan species but greater G0F and G2F glycan species as well as higheramounts of acidic charge variants (FIG. 11).

III. Addition of Ethanol Using Microscale Bioreactors for Production ofa Different Monoclonal Antibody

Materials and Methods:

Antibody production was performed as described above for mAb 2. Thiscell line is a CHO.DG44 derived clone producing mAb 2.

Ethanol was fed daily between 0.0% and 0.15% v/v. Calculated cumulativecell culture concentration (concentration in vessel at time of cellharvest) of ethanol ranged between 0.0%-1.35% v/v. Bioreactor physicalconditions were maintained at a pH of 7.0±0.1 shifted to 6.8±0.1,dissolved oxygen maintained at 30% air saturation and a constanttemperature of 35° C. Basal and feed media were fully synthetic andchemically defined containing no animal derived components.

Results

As shown in FIG. 12, higher ethanol concentrations are associated withdecreased Man5 and G0F glycan species, increased G0, G1F, and G2F glycanspecies, and decreased high molecular weight species.

Ethanol supplementation altered glycosylation profile, antibodyaggregates and acidic and basic charge variants. These effects sometimesvaried among varying cell lines, mAb products and/or feed formulations.

In most cases ethanol decreased Man5%, G0% and G0F % and increased themature glycan species G1F % and G2F %.

Example 3: Addition of Fatty Acids to Cell Culture Medium ImprovesProduction of Recombinant Polypeptides

Exogenous fatty acid supplementation could potentially decreaseintracellular energy consumption since adenosine triphosphate (ATP) isconsumed in the conversion of acetyl-CoA to malonyl-CoA, the primarybuilding block of intracellular fatty acids. Furthermore, fatty acidsupplementation possibly strengthens membrane integrity as the majorityof cellular membranes are composed of C16 and C18 length phospholipids.

I. Determination of Cytotoxicity

Fatty acid cytotoxicity to CHO.DXB11 cells was determined by daily feeddosing. 4 mL of CHO cells at 4 e⁶/mL cellular density were seeded into 6well deep plates, mixed at 150 RPM, and daily fed fatty acids. Fattyacids were solubilized in ethanol and fed once daily to increase wellplate concentration from 10 μM to 160 μM. Cell growth rate and cellviability were negatively affected at varying levels—dependent on theindividual fatty acid. Oleic acid, linoleic acid and linolenic acidbecame toxic around 40 μM daily feeding concentration. Myristic acid,palmitic acid and stearic acid became toxic around 80 μM daily feedingconcentration. Cholesterol started to become toxic around 50 μM dailyfeeding concentration.

II. Addition of Oleic Acid Using Bench-Scale Bioreactors

Materials and Methods:

Antibody production was performed as described above for mAb 1.

Feed media was supplemented with fatty acids to determine if fatty acidsaltered mAb properties. Feed media was fed daily in a fed-batch processusing bench-scale (2 L working volume) bioreactors. Oleic acid was feddaily to increase working bioreactor concentration by 40 μM. Calculatedcumulative cell culture concentration (concentration in vessel at timeof cell harvest) of oleic acid was 440 μM. The media used for baselinemeasurements did not contain any fatty acids or ethanol.

Bioreactor physical conditions were maintained at a pH of 7.0±0.2,dissolved oxygen at 30% air saturation, temperature of 35° C. duringlogarithmic cellular growth phase and shifted to 29° C. during cellularproduction phase (temperature was shifted one day before max viable celldensity was reached). Basal and feed media were fully synthetic andchemically defined containing no animal derived components.

Results:

As shown in FIG. 13, addition of oleic acid to media increasedmonoclonal antibody titer, decreased Man5, G0, and G0F glycan species,increased G1F and G2F glycan species, decreased high molecular weightspecies, and decreased acidic charge variants.

Addition of oleic acid using bench-scale bioreactors using bench-scalebioreactors with different physical and feed conditions

Materials and Methods:

Antibody production was performed as described above for mAb 1.

Ethanol supplemented media was further supplemented with fatty acids todetermine if fatty acids altered mAb properties. Feed media was feddaily in a fed-batch process using bench-scale (2 L working volume)bioreactors. Oleic acid was fed daily to increase working bioreactorconcentration by 40 μM. Calculated cumulative cell culture concentration(concentration in vessel at time of cell harvest) of oleic acid was 440μM. Media used for baseline measurements contains ethanol but no fattyacids.

Bioreactor physical conditions were maintained at a pH of 7.0±0.2,dissolved oxygen at 30% air saturation, temperature of 35° C. duringlogarithmic cellular growth phase and shifted to 31° C. during cellularproduction phase (temperature was shifted one day before max viable celldensity was reached). Basal and feed media are fully synthetic andchemically defined containing no animal derived components.

Results:

As shown in FIG. 14, addition of oleic acid resulted in decreasedantibody titer, decreased Man5, G0, GIF, and G2F glycan species,increased G0F glycan species, increased high and low molecular weightspecies, decreased acidic charge variants, and increased basic chargevariants. Oleic acid supplementation appeared to further reduce Man5%levels from that of just ethanol supplementation. It additionallyaltered fucosylated glycan species and reduced acidic charge variants.

Overall, oleic acid dissolved in ethanol was observed to increase G0F %glycan profile. Supplementation appeared to reduce Man5%, G0%, G1F %,G2F % glycan profile in addition to reducing acidic charge species.

Example 4: Addition of Sodium Chloride to Medium for Comparison toAddition of Lithium Chloride

Sodium concentration in a normal cellular interstitial environment istypically 136-145 mM and chloride is typically 96-106 mM. For thelithium supplemented medium described above, approximately 1 mM lithiumchloride is added daily, which is roughly a 1% increase in chloride ionconcentration. Accordingly, this Example shows that the improvement incell productivity results from increased lithium ion concentrationrather than increased amounts of chloride ion.

Materials and Methods:

Monoclonal antibody m Ab3) is a monoclonal antibody produced usingrecombinant DNA technology. The expression vector is fully syntheticwith both heavy- and light-chain gene sequences regulated by strongconstitutive promoters. Gene sequences were confirmed by DNA sequencing.The expression vector was linearized by restriction enzymes and stablytransfected into CHO.DXB11 cells by electroporation. Gene amplificationsteps were performed with increasing concentrations of methotrexate.Single cell cloning was performed after gene amplification. Selectsingle cell clones were expanded and cryopreserved. The cell line usedwas a CHO.DXB11 derived clone.

Sodium chloride was fed daily at 4.59 mM and 9.18 mM using two differentprocess strategies. Process strategy 1 was maintained at 36.5° C. thenshifted to 31° C. during production phase. Process strategy 2 wasmaintained at 36.5° C. then shifted to 31° C. during production phasethen re-shifted back to 36.5° C. during late production phase. Bothprocess strategies were maintained at 30% air saturation and a pH of7.0±0.2. Other glycan species and HMW % and LMW % are not listed becauseno significant change in levels were observed from sodium chloridesupplementation.

Results:

As shown in FIG. 15A and FIG. 15B, addition of sodium chloride to mediadecreased monoclonal antibody titer and G0F glycan species. Man5, G0,G1F and G2F glycan species increased with acidic and basic chargevariants.

When supplementing increased levels of sodium chloride, mAb propertieswere altered and significantly different than when supplemented withlithium chloride. Sodium chloride reduced titer levels, alteredglycosylation profile differently, increased basic % and did not changeLMW % or HMW %. The data indicate that the effects observed with lithiumchloride are due to the lithium ion and not chloride.

Example 5: Supplementation of Media with Fatty Acids, Methyl Esters,Sterols, Glycerides and Isoprenoids

A variety of fatty acids, methyl esters, sterols, glycerides andisoprenoids were added to cell culture media used to produce twoseparate monoclonal antibodies followed by evaluation of titer andproduct quality.

Materials and Methods:

Monoclonal antibodies 1 (mAb1) and 4 (mAb4) were produced using twoseparate cell lines according to methods described in the previousExamples. Titer, percent of high molecular weight and low molecularweight species, acidic and basic charge variants, and glycosylationspecies profiles were assessed as in the previous Examples.

Fatty acids were dissolved in ethanol and fed daily according to Table 5using two different process strategies for mAb1 and mAb4. The mAb1process strategy was maintained at 37° C. then shifted to 31° C. duringlate exponential phase, with the pH being maintained at 7.0±0.2throughout the culture. The mAb4 process strategy was maintained at36.5° C. then shifted to 33° C. during production phase, with the pHbeing shifted from 7.0±0.1 to 6.8±0.1 in the late exponential phase.Both process strategies were maintained at 30% air saturation

TABLE 5 Feed conditions used for cell culture. mAb1 Feed Conditions mAb4Feed Conditions Daily Fatty Daily Daily Fatty Daily Acid Feed EthanolAcid Feed Ethanol Supplement (μM) Feed (% v/v) (μM) Feed (% v/v) Control0.0 0.05 0.0 0.09 Thymol 2.7 0.05 5.0 0.09 Cholesteryl 10.7 0.05 20.00.09 acetate Methyl 2.7 0.05 5.0 0.09 octanoate 1-Octanoyl- 2.7 0.05 5.00.09 rac-glycerol Oleic Acid 10.7 0.05 20.0 0.09 Linoleic acid 10.7 0.0520.0 0.09 Linolenic acid 10.7 0.05 20.0 0.09 Cholesterol 7.4 0.05 20.00.09 Palmitic Acid 10.7 0.05 20.0 0.09 Stearic acid 10.7 0.05 20.0 0.09Myristic acid 10.7 0.05 20.0 0.09

Results

As shown in FIG. 16, media supplementation with thymol,1-octanoyl-Rac-glycerol, linoleic acid, and linolenic acid increased thetiter of mAb1 while linolenic acid increased production of mAb4 Withrespect to HMW species, palmitic acid decreased the relative percentageversus controls for mAb1 (FIG. 17A) whereas thymol, cholesterol acetate,methyl octanoate, 1-octanoyl-rac-glycerol, palmitic acid, stearic acid,and myristic acid decreased the relative percentage of BMW species formAb4 (FIG. 17A). Low molecular weight species were decreased for mAb1using thymol, 1-octanoyl-rac-glycerol, linoleic acid, linolenic acid,palmitic acid, and stearic acid (FIG. 17B) whereas thymol, cholesterylacetate, linoleic acid, and palmitic acid decreased LMW species for mAb4(FIG. 17B).

Acidic charge variants were decreased for mAb1 production viasupplementation with thymol, methyl octanoate, 1-octanoyl-rac-glycerol,oleic acid, linoleic acid, linolenic acid, palmitic acid, and myristicacid (FIG. 18A) whereas thymol, cholesteryl acetate,1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid,palmitic acid, stearic acid, and myristic acid decreased acidic chargevariants for mAb4 (FIG. 18A). Media supplementation with cholesterol,palmitic acid, and stearic acid decreased basic charge variants for mAb1production (FIG. 18B) while thymol, cholesteryl acetate, methyloctanoate, 1-octanoyl-rac-glycerol, cholesterol, palmitic acid, stearicacid, and myristic acid decreased the relative percentage of basiccharge variants for mAb4 (FIG. 18B).

Decreased Man5 glycan species were observed following mediasupplementation with cholesteryl acetate for mAb1 production (FIG. 19A)while supplementation with thymol, cholesteryl acetate, methyloctanoate, 1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenicacid, cholesterol, palmitic acid, stearic acid, and myristic aciddecreased the relative percentage of Man5 glycan species for mAb4production (FIG. 19A). Decreased G0 glycan species for mAb1 wereobserved following media supplementation with thymol, cholesterylacetate, methyl octanoate, oleic acid, linolenic acid, cholesterol,palmitic acid, stearic acid, and myristic acid (FIG. 19B) whereassupplementation with cholesteryl acetate, methyl octanoate,1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid,cholesterol, palmitic acid, stearic acid, and myristic acid decreasedthe relative percentage of G0 glycan species for mAb4 production (FIG.19B). Finally, decreased G0F glycan species were observed for mAb1following media supplementation with cholesteryl acetate, methyloctanoate, 1-octanoyl-rac-glycerol, oleic acid, and myristic acid (FIG.20) whereas supplementation with 1-octanoyl-rac-glycerol decreased therelative percentage of G0F glycan species for mAb4 production (FIG. 20).

1. A method for producing one or more recombinant polypeptides from anengineered mammalian cell, the method comprising: (a) culturing saidengineered mammalian cell in a medium under suitable conditions for theproduction of said one or more recombinant polypeptides; and (b)producing said one or more recombinant polypeptides, wherein the mediumcomprises (i) a basal medium or a feed medium; and (ii) ethanol.
 2. Themethod of claim 1, wherein the method further comprises (c) isolatingsaid one or more recombinant polypeptides.
 3. The method of claim 1,wherein the medium is (a) a basal medium; or (b) a feed medium.
 4. Themethod of claim 1, wherein said one or more recombinant polypeptides isan antibody or fragment thereof.
 5. The method of claim 4, wherein saidantibody is a monoclonal antibody.
 6. The method of claim 5, wherein themonoclonal antibody inhibits the growth of a proliferating cell.
 7. Themethod of claim 4, wherein said antibody or fragment thereof binds toHER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR,respiratory syncytial virus (RSV), glycoprotein IIb/IIIa, IgG1, IgE,complement component 5 (C5), B-cell activating factor (BAFF), CD19,CD30, interleukin-1 beta (IL1β), prostate specific membrane antigen(PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertasesubtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7.
 8. The method of claim 5, wherein said monoclonalantibody is trastuzumab, pertuzumab, infliximab, adalimumab,bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab,daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.
 9. A method for modulating theglycosylation profile of one or more recombinant polypeptides producedby a genetically engineered mammalian cell, the method comprising: (a)culturing said mammalian cell in a medium under suitable conditions forthe production of said one or more recombinant polypeptides; and (b)producing said one or more recombinant polypeptides, wherein the mediumcomprises (i) a basal medium or a feed medium; and (ii) ethanol, andwherein said one or more recombinant polypeptides has a modulatedglycosylation profile compared to recombinant polypeptides produced bymammalian cells that are not cultured in the medium.
 10. The method ofclaim 9, wherein said modulated glycosylation profile comprisesmodulated terminal mannose glycan species.
 11. The method of claim 9,wherein said modulated glycosylation comprises modulation of one or moreglycan species selected from mannose-5-N-acetylglycosamine-2 (Man5),mannose-6-N-acetylglycosamine-2 (Man6), mannose-3-N-acetylglucosamine-4(G0), mannose-3-N-acetylglucosamine-4-fucose (G0F),mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), and/ormannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F).
 12. The methodof claim 9, wherein the ratio of the terminal mannose glycan species tothe total sum of glycan species is modulated by about 40% to about 50%.13. The method of claim 9, wherein said one or more recombinantpolypeptides is an antibody or fragment thereof.
 14. The method of claim13, wherein said antibody is a monoclonal antibody.
 15. The method ofclaim 14, wherein the monoclonal antibody inhibits the growth of aproliferating cell.
 16. The method of claim 13, wherein said antibody orfragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52,CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoproteinIIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activatingfactor (BAFF), CD19, CD30, interleukin-1 beta 441), prostate specificmembrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proproteinconvertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7.
 17. The method of claim 14, wherein said monoclonalantibody is trastuzumab, pertuzumab, infliximab, adalimumab,bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab,daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.
 18. A method for modulating theamount of high or low molecular weight species of one or morerecombinant polypeptides produced by an engineered mammalian cell, themethod comprising: (a) culturing said mammalian cell in a medium undersuitable conditions for the production of said one or more recombinantpolypeptides; and (b) producing said one or more recombinantpolypeptides, wherein the medium comprises (i) a basal medium or a feedmedium; and (ii) ethanol, and wherein said one or more recombinantpolypeptides have reduced amounts of high or low molecular weightspecies compared to recombinant polypeptides produced by mammalian cellsthat are not cultured in the medium.
 19. The method of claim 18, whereinsaid one or more recombinant polypeptides has reduced amounts of (a)high molecular weight species; or (b) low molecular weight species. 20.The method of claim 19, wherein said low molecular weight speciescomprise polypeptide fragments that are not completely assembled and/orfolded.
 21. The method of claim 19, wherein said high molecular weightspecies comprise more than one subunit of a recombinant polypeptide. 22.The method of claim 21, wherein the percent specific ratio of lowmolecular weight species to the sum of all (1) non-aggregated; (2) lowmolecular weight species; and (3) high molecular weight species ismodulated relative to the percent specific ratio compared to recombinantpolypeptides produced by mammalian cells that are not cultured in themedium
 23. The method of claim 22, wherein the percent specific ratio ofhigh molecular weight species to the sum of all (1) non-aggregated; (2)low molecular weight species; and (3) high molecular weight species ismodulated relative to the percent specific ratio compared to recombinantpolypeptides produced by mammalian cells that are not cultured in themedium.
 24. The method of claim 18, wherein said one or more recombinantpolypeptides is an antibody or fragment thereof.
 25. The method of claim24, wherein said antibody is a monoclonal antibody.
 26. The method ofclaim 25, wherein the monoclonal antibody inhibits the growth of aproliferating cell.
 27. The method of claim 24, wherein said antibody orfragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52,CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoproteinIIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activatingfactor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specificmembrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proproteinconvertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7.
 28. The method of claim 25, wherein said monoclonalantibody is trastuzumab, pertuzumab, infliximab, adalimumab,bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab,daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.
 29. A method for modulating theamount of acidic or basic charge species of one or more recombinantpolypeptides produced by an engineered mammalian cell, the methodcomprising: (a) culturing said mammalian cell in a medium under suitableconditions for the production of said one or more recombinantpolypeptides; and (b) producing said one or more recombinantpolypeptides, wherein the medium comprises (i) a basal medium or a feedmedium; and (ii) ethanol, and wherein said one or more recombinantpolypeptides have reduced amounts of acidic charge species compared torecombinant polypeptides produced by mammalian cells that are notcultured in the medium.
 30. The method of claim 29, wherein the percentspecific ratio of acidic or basic charge species to the total sum of all(1) acidic species; (2) main species; and (3) basic charge species isreduced relative to recombinant polypeptides produced by mammalian cellsthat are not cultured in the medium.
 31. The method claim 29, whereinsaid one or more recombinant polypeptides is an antibody or fragmentthereof.
 32. The method of claim 31, wherein said antibody is amonoclonal antibody.
 33. The method of claim 31, wherein said antibodyor fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20,CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoproteinIIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activatingfactor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specificmembrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proproteinconvertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxicT-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5),programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen(PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6),interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23(IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-likekinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin,oxidized low-density lipoprotein (oxLDL), platelet-derived growth factorreceptor beta, neuropilin 1, Von Willebrand factor (vWF), integrinα_(V)β₃, neural apoptosis-regulated proteinase 1, integrin α_(IIb)β₃,beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A),nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, orintegrin α4β7.
 34. The method of claim 32, wherein said monoclonalantibody is trastuzumab, pertuzumab, infliximab, adalimumab,bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab,daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab,palivizumab, abciximab, basiliximab, ibritumomab, omalizumab,eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab,brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab,eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab,mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab,palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab,siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab,romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab,evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab,ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab,demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab,aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab,bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab,solanezumab, tanezumab, and vedolizumab.
 35. The method of claim 1,wherein ethanol is present at a concentration from about 0.001% to about4% (v/v).
 36. The method of claim 1, wherein the medium furthercomprises (c) one or more fatty acids; and/or (d) one or more sources oflithium ions.
 37. The method of claim 36, wherein said one or more fattyacids is selected from the group consisting of oleic acid, linoleicacid, linolenic acid, myristic acid, palmitic acid stearic acid, thymol,cholesteryl acetate, methyl octanoate, 1-octanoyl-rac-glycerol,cholesterol, butyric (C4), valeric (C5), caproic (C6), enanthic (C7),caprylic (C8), pelargonic (C9), capric (C10), undecylic (C11), lauric(C12), tridecylic (C13), myristic (C14), pentadecanoic (C15), margaric(C17), nonadecylic (C19), arachidic (C20), heneicosylic (C21), behenic(C22), tricosylic (C23), lignoceric (C24), pentacosylic (C25), cerotic(C26), heptacosylic (C27), montanic (C28), nonacosylic (C29), melissic(C30), hentriacontylic (C31), lacceroic (C32), psyllic (C33), geddic(C34), ceroplastic (C35), hexatriacontylic (C36), heptatriacontanoic(C37), and octatriacontanoic (C38) acids.
 38. The method of claim 36,wherein the one or more fatty acids are present at a concentration ofabout 1 μM to about 4 mM.
 39. The method of claim 36, wherein said oneor more sources of lithium ions is selected from the group of one ormore of lithium acetate, lithium chloride, lithium carbonate, lithiumoxybutyrate, lithium orotate, lithium bromide, lithium citrate, lithiumfluoride, lithium iodide, lithium nitrate, and lithium sulfate.
 40. Themethod of claim 36, wherein said lithium ions are present in aconcentration from about 0.1 μM to about 25 mM.
 41. The method of claim1, wherein the titer of said one or more recombinant polypeptides isincreased compared to the titer of recombinant polypeptides produced bymammalian cells that are not cultured in said medium.
 42. The method ofclaim 1, wherein the amount of high molecular weight species of said oneor more recombinant polypeptides produced by said cells is modulatedcompared to recombinant polypeptides produced by mammalian cells thatare not cultured in said medium.
 43. The method of claim 1, wherein theamount of low molecular weight species of said one or more recombinantpolypeptides produced by said cells is modulated compared to recombinantpolypeptides produced by mammalian cells that are not cultured in saidmedium.